( E ) Representative flicker traces at three months of age show the rescue of flicker responses of NSC668394 - treated miR - 211 - / - mice ( green lines ) compared to DMSO - treated miR - 211 - / - control mice ( red lines ) . WT mice were used as a control ( black lines ) . Flicker recordings were performed with light intensities ranging from 10 − 4 to 15 cd s / m2 in steps of 0 . 6 logarithmic units at 6 Hz frequency . ( F ) Flicker responses , plotted as a function of stimulus intensity , from WT ( black lines ) , DMSO - treated miR - 211 - / - ( red lines ) and NSC668394 - treated miR - 211 - / - ( green lines ) mice , at three months of age . The amplitude of the recordings from NSC668394 miR - 211 - / - treated mice is significantly rescued compared to DMSO - treated miR - 211 - / - mice . WT mice were used as a control . Error bars represent s . e . m . ANOVA test ( DMSO vs WT and NSC668394 vs DMSO treated mice ) * * p ≤ 0 . 01 , * * * p ≤ 0 . 005 .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O

(E) Representative flicker traces at three months of age show the rescue of flicker responses of NSC668394-treated miR-211-/- mice (green lines) compared to DMSO-treated miR-211-/- control mice (red lines). WT mice were used as a control (black lines). Flicker recordings were performed with light intensities ranging from 10−4 to 15 cd s/m2 in steps of 0.6 logarithmic units at 6 Hz frequency. (F) Flicker responses, plotted as a function of stimulus intensity, from WT (black lines), DMSO-treated miR-211-/- (red lines) and NSC668394-treated miR-211-/- (green lines) mice, at three months of age. The amplitude of the recordings from NSC668394 miR-211-/- treated mice is significantly rescued compared to DMSO-treated miR-211-/- mice. WT mice were used as a control. Error bars represent s.e.m. ANOVA test (DMSO vs WT and NSC668394 vs DMSO treated mice) **p ≤ 0.01, ***p ≤ 0.005.


. A representative example of a 7 days ( 7d ) old adult female posterior midgut at low ( A ) and high magnification ( B , Region 4 - Region 5 ) showing the initiator caspase reporter DBS - S - QF ( Red , immunostaining anti - HA ) . Flies were reared under experimental regime that protects the intestinal epithelial integrity ( intestine reared in Oxford Medium and flies transferred every two days into vials with fresh food , see Figure EV1 ) at 25 Co . The image on the left was acquired at low magnification with the 10x objective , while the right panel was acquired using the 40x . The left and right images in all examples correspond to independent intestines of the same genotype and treatment . Genotype : y1 w1118 UAS - mCD8 : : GFP . L QUAS - mtdTomato - 3xHA Act - DBS - S - QF ( BL83131 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

. A representative example of a 7 days (7d) old adult female posterior midgut at low (A) and high magnification (B, Region 4-Region 5) showing the initiator caspase reporter DBS-S-QF (Red, immunostaining anti-HA). Flies were reared under experimental regime that protects the intestinal epithelial integrity (intestine reared in Oxford Medium and flies transferred every two days into vials with fresh food, see Figure EV1) at 25 Co. The image on the left was acquired at low magnification with the 10x objective, while the right panel was acquired using the 40x. The left and right images in all examples correspond to independent intestines of the same genotype and treatment. Genotype: y1 w1118 UAS-mCD8::GFP.L QUAS-mtdTomato-3xHA Act-DBS-S-QF (BL83131)


B . A representative example of a 7 d intestine reared at 29 Co in experimental conditions that protect the epithelial integrity and labelled with ReDDM cell - lineage tool . esg ( green cytoplasmic signal ) labels intestinal progenitor cells , whilst Histone - RFP ( red nuclear signal ) acts as a semi - permanent marker that allows the visualization of differentiated cells . Note the extensive overlap between the two markers and the absence of differentiated cells only showing the Histone - RFP labelling , as an indication of negligible epithelial turnover . The image on the left was acquired at low magnification with the 10x objective , while the right panel was acquired using the 40x . White dotted line outlines the gut using DAPI staining ( not shown ) as a reference . Genotype : w1118 ; esg - Gal4 UAS - CD8 - GFP ( Irene Miguel Aliaga ) / Cyo ; UAS - Histone - RFP ( BL7019 ) TubG80ts ( BL7019 C . Quantification of intestinal cell subpopulations labelled with ReDDM system at high magnification ( GFP and Histone - RFP ) and different time points post ReDDM activation ( 3 - and 7 - days ) in experimental conditions that protect the epithelial integrity ; note that none of the cell populations in the gut ( GFP ( n . s . no significant , P = 0 . 5267 ) or Histone - RFP ( n . s . , P = 0 . 2752 ) significantly increase in number overtime ( Quantifications were made using N ≥ 2 biological replicates , total number of guts analysed at 3 days n = 34 and at 7 days n = 45 ; Mann - Whitney , C ) . Error bars represent Standard Error of the Mean . Genotype : w1118 ; esg - Gal4 UAS - CD8 - GFP ( Irene Miguel Aliaga ) / Cyo ; UAS - Histone - RFP ( BL7019 ) TubG80ts ( BL7019 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. A representative example of a 7 d intestine reared at 29 Co in experimental conditions that protect the epithelial integrity and labelled with ReDDM cell-lineage tool. esg (green cytoplasmic signal) labels intestinal progenitor cells, whilst Histone-RFP (red nuclear signal) acts as a semi-permanent marker that allows the visualization of differentiated cells. Note the extensive overlap between the two markers and the absence of differentiated cells only showing the Histone-RFP labelling, as an indication of negligible epithelial turnover. The image on the left was acquired at low magnification with the 10x objective, while the right panel was acquired using the 40x. White dotted line outlines the gut using DAPI staining (not shown) as a reference. Genotype: w1118; esg-Gal4 UAS-CD8-GFP (Irene Miguel Aliaga)/ Cyo; UAS-Histone-RFP (BL7019) TubG80ts (BL7019 C. Quantification of intestinal cell subpopulations labelled with ReDDM system at high magnification (GFP and Histone-RFP) and different time points post ReDDM activation (3- and 7-days) in experimental conditions that protect the epithelial integrity; note that none of the cell populations in the gut (GFP (n.s. no significant , P = 0.5267) or Histone-RFP (n.s. , P =0.2752) significantly increase in number overtime (Quantifications were made using N ≥ 2 biological replicates, total number of guts analysed at 3 days n =34 and at 7 days n=45; Mann-Whitney, C). Error bars represent Standard Error of the Mean. Genotype: w1118; esg-Gal4 UAS-CD8-GFP (Irene Miguel Aliaga)/ Cyo; UAS-Histone-RFP (BL7019) TubG80ts (BL7019)


D . A representative example of an adult female posterior midgut at low ( A ) and high magnification ( B , Region 4 - Region 5 ) showing the initiator caspase reporter DBS - S - QF ( Red , immunostaining anti - HA ) after 16 hours of paraquat treatment in Oxford food at 25 Co ; note the expansion of the labelling with DBS - S - QF to large intestinal cells ( ECs ) ( compare D with A ) . The image on the left was acquired at low magnification with the 10x objective , while the right panel was acquired using the 40x . White dotted line outlines the gut using DAPI staining as a reference . Genotype : y1 w1118 UAS - mCD8 : : GFP . L QUAS - mtdTomato - 3xHA Act - DBS - S - QF ( BL83131 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. A representative example of an adult female posterior midgut at low (A) and high magnification (B, Region 4-Region 5) showing the initiator caspase reporter DBS-S-QF (Red, immunostaining anti-HA) after 16 hours of paraquat treatment in Oxford food at 25 Co; note the expansion of the labelling with DBS-S-QF to large intestinal cells (ECs) (compare D with A). The image on the left was acquired at low magnification with the 10x objective, while the right panel was acquired using the 40x. White dotted line outlines the gut using DAPI staining as a reference. Genotype: y1 w1118 UAS-mCD8::GFP.L QUAS-mtdTomato-3xHA Act-DBS-S-QF (BL83131)


E . ReDDM lineage - tracing in an adult intestine reared in Oxford Medium and paraquat ( 20 mM ) during 16 hours at 29 Co ; note the abundance of Histone - RFP cells without GFP signal , as an indication of epithelial damage and subsequent differentiation of progenitor cells . The image on the left was acquired at low magnification with the 10x objective , while the right panel was acquired using the 40x . Genotype : w1118 ; esg - Gal4 UAS - CD8 - GFP ( Irene Miguel Aliaga ) / Cyo ; UAS - Histone - RFP ( BL7019 ) TubG80ts ( BL7019 ) F . Quantification of ReDDM labelling at high magnification after paraquat treatment ; note the statistically significant increase ( * * ; P = 0 . 0099 ) of Histone - RFP expressing cells without GFP signal ( Quantifications were made using N ≥ 2 biological replicates ; Unpaired two - tailed t test , 3d n = 9 , 7d n = 7 ) . Error bars represent Standard Error of the Mean . Genotype : w1118 ; esg - Gal4 UAS - CD8 - GFP ( Irene Miguel Aliaga ) / Cyo ; UAS - Histone - RFP ( BL7019 ) TubG80ts ( BL7019 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. ReDDM lineage-tracing in an adult intestine reared in Oxford Medium and paraquat (20 mM) during 16 hours at 29 Co; note the abundance of Histone-RFP cells without GFP signal, as an indication of epithelial damage and subsequent differentiation of progenitor cells. The image on the left was acquired at low magnification with the 10x objective, while the right panel was acquired using the 40x. Genotype: w1118; esg-Gal4 UAS-CD8-GFP (Irene Miguel Aliaga)/ Cyo; UAS-Histone-RFP (BL7019) TubG80ts (BL7019) F. Quantification of ReDDM labelling at high magnification after paraquat treatment; note the statistically significant increase (**; P = 0.0099) of Histone-RFP expressing cells without GFP signal (Quantifications were made using N ≥ 2 biological replicates; Unpaired two-tailed t test, 3d n = 9, 7d n =7). Error bars represent Standard Error of the Mean. Genotype: w1118; esg-Gal4 UAS-CD8-GFP (Irene Miguel Aliaga)/ Cyo; UAS-Histone-RFP (BL7019) TubG80ts (BL7019)


H . Representative example of an intestine 7d old expressing two copies of the effector caspase inhibitor P35 under the regulation of esg - gal4 ( green immunostaining with antibody against P35 ) at 29 Co . Genotype : w1118 ; esg - Gal4 UAS - CD8 - GFP / UAS - P35 ( BL5072 ) ; UAS - P35 ( BL5073 ) / + I . ReDDM lineage - tracing system in a Drosophila intestine expressing two copies of the effector caspase inhibitor P35 under the regulation of esg - Gal4 and experimental conditions that protect the epithelial integrity at 29 Co . Genotype : w1118 ; esg - Gal4 UAS - CD8 - GFP / UAS - P35 ( BL5072 ) ; UAS - Histone - RFP TubG80ts / UAS - P35 ( BL5073 ) J . ReDDM quantification corresponding to the intestines described in ( I ) ; no significant increase in either esg ( n . s . , P = 0 . 1352 ) or Histone - RFP ( n . s . , P = 0 . 9801 ) cell number is observed ( Quantifications were made using N ≥ 2 biological replicates ; unpaired two - tailed t test , 3d n = 12 , 7d n = 11 ) . Error bars represent Standard Error of the Mean . DAPI ( blue ) labels the nuclei in panels A , D and H . Genotype : w1118 ; esg - Gal4 UAS - CD8 - GFP / UAS - P35 ( BL5072 ) ; UAS - Histone - RFP TubG80ts / UAS - P35 ( BL5073 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H. Representative example of an intestine 7d old expressing two copies of the effector caspase inhibitor P35 under the regulation of esg-gal4 (green immunostaining with antibody against P35) at 29 Co. Genotype: w1118; esg-Gal4 UAS-CD8-GFP / UAS-P35 (BL5072); UAS-P35 (BL5073)/+ I. ReDDM lineage-tracing system in a Drosophila intestine expressing two copies of the effector caspase inhibitor P35 under the regulation of esg-Gal4 and experimental conditions that protect the epithelial integrity at 29 Co. Genotype: w1118; esg-Gal4 UAS-CD8-GFP/ UAS-P35 (BL5072); UAS-Histone-RFP TubG80ts / UAS-P35 (BL5073) J. ReDDM quantification corresponding to the intestines described in (I); no significant increase in either esg (n.s. , P = 0.1352) or Histone-RFP (n.s. , P = 0.9801) cell number is observed (Quantifications were made using N ≥ 2 biological replicates; unpaired two-tailed t test, 3d n = 12, 7d n = 11). Error bars represent Standard Error of the Mean. DAPI (blue) labels the nuclei in panels A, D and H. Genotype: w1118; esg-Gal4 UAS-CD8-GFP/ UAS-P35 (BL5072); UAS-Histone-RFP TubG80ts / UAS-P35 (BL5073)


A . ReDDM activation in Dronc heterozygous ( + / - ) intestine 7 days after temperature shift at 29 Co ; esg expression ( green ) labels the intestinal progenitor cells , Histone - RFP ( red ) is a semi - permanent marker retained in differentiated cells and Pdm - 1 ( grey ) labels differentiated ECs . The red arrows indicates the enlarged areas depicted in the insets in the entire figure . DAPI labels the DNA In the entire Figure ( blue ) . All of the experiments in the entire figure have been made under experimental conditions that protect the epithelial integrity ( intestine reared in Oxford Medium and flies transferred every two days into vials with fresh food ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. ReDDM activation in Dronc heterozygous (+/-) intestine 7 days after temperature shift at 29 Co; esg expression (green) labels the intestinal progenitor cells, Histone-RFP (red) is a semi-permanent marker retained in differentiated cells and Pdm-1 (grey) labels differentiated ECs. The red arrows indicates the enlarged areas depicted in the insets in the entire figure. DAPI labels the DNA In the entire Figure (blue). All of the experiments in the entire figure have been made under experimental conditions that protect the epithelial integrity (intestine reared in Oxford Medium and flies transferred every two days into vials with fresh food).


B . ReDDM activation in a Drosophila intestine in which all of the progenitor cells are fully mutant for Dronc ( see full genotype in methods ) ; note the increased cell size of esg - expressing cells ( GFP + , green ) , the co - expression of Histone - RFP ( red ) and the EC marker Pdm - 1 ( gray ) , and the coexpression of Pdm - 1 and GFP in enlarged cells ( compare panels and detailed insets from A with B ) . The image is a representative example of a Drosophila adult gut 7 days after temperature shift at 29 Co . Genotype : w1118 ; esg - Gal4 UAS - CD8 - GFP / + ; TubG80ts UAS - Histone - RFP DroncKO / UAS - Flippase ( BL8209 ) FRT Dronc - GFP - APEX FRT suntag - HA -

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. ReDDM activation in a Drosophila intestine in which all of the progenitor cells are fully mutant for Dronc (see full genotype in methods); note the increased cell size of esg-expressing cells (GFP+, green), the co-expression of Histone-RFP (red) and the EC marker Pdm-1 (gray), and the coexpression of Pdm-1 and GFP in enlarged cells (compare panels and detailed insets from A with B). The image is a representative example of a Drosophila adult gut 7 days after temperature shift at 29 Co. Genotype: w1118; esg-Gal4 UAS-CD8-GFP / + ; TubG80ts UAS-Histone-RFP DroncKO / UAS-Flippase (BL8209) FRT Dronc-GFP-APEX FRT suntag-HA-Cherry


C . Relative number of esg - expressing cells normalised to DAPI ; notice that the relative percentage of esg - labelled cells is significantly higher in Dronc fully mutant conditions ( - / - ) at 3 days ( * * * ; P = 0 . 0007 ) and 7d ( * * * * ; p < 0 . 0001 ) post temperature shift at 29 Co ( Quantifications were made using N ≥ 2 biological replicates ; unpaired two - tailed t - test , + / - n = 32 , - / - n = 25 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. Relative number of esg-expressing cells normalised to DAPI; notice that the relative percentage of esg-labelled cells is significantly higher in Dronc fully mutant conditions (-/-) at 3 days (***; P = 0.0007) and 7d (****; p <0.0001) post temperature shift at 29 Co (Quantifications were made using N ≥ 2 biological replicates; unpaired two-tailed t-test, +/- n=32, -/- n=25).


D . Average cell size of esg - expressing cells ( μm² ) ; note the increased cell size of Dronc fully mutant progenitor cells ( - / - ) at 3d ( * * ; P = 0 . 0014 ) and 7d ( * * * , P = 0 . 0007 ) post temperature shift at 29 Co ( Quantifications were made using N ≥ 2 biological replicates ; unpaired two - tailed parametric t - test , 3d + / - n = 19 , 3d - / - n = 28 , 7d + / - n = 29 , 7d - / - n = 28 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. Average cell size of esg-expressing cells (μm²); note the increased cell size of Dronc fully mutant progenitor cells (-/-) at 3d (**; P = 0.0014) and 7d (***, P = 0.0007) post temperature shift at 29 Co (Quantifications were made using N ≥ 2 biological replicates; unpaired two-tailed parametric t-test, 3d +/- n = 19, 3d -/- n = 28, 7d +/- n = 29, 7d -/- n = 28).


E . Relative number of esg - negative cells expressing Histone - RFP normalised to DAPI ; notice that the number of Histone - RFP cells without esg expression is significantly higher in Dronc fully mutant ( - / - ) conditions at 7d ( * ; P = 0 . 0046 ) ( Quantifications were made using N biological replicates ≥ 2 ; Mann - Whitney test , + / - n = 24 , - / - n = 20 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. Relative number of esg-negative cells expressing Histone-RFP normalised to DAPI; notice that the number of Histone-RFP cells without esg expression is significantly higher in Dronc fully mutant (-/-) conditions at 7d (*; P = 0.0046) (Quantifications were made using N biological replicates ≥ 2; Mann-Whitney test, +/- n = 24, -/- n = 20.


. A 7d intestine Dronc heterozygous ( + / - ) intestine in which intestinal stem cells express GFP under the regulation of Dl - Gal4 . All of the experiments described in the figure were performed in Oxford medium following an experimental regime that protects the epithelial integrity ( flies transferred every two days into vials with fresh food ) . DAPI ( blue ) labels the DNA in the entire Figure . Genotype : w1118 ; ; delta - Gal4 UAS - GFP TubG80ts UAS - Histone - RFP DroncKO / + ( recombinant chromosome made for this study ; the parental line delta - Gal4 UAS - GFP TubG80ts UAS - Histone - RFP was obtained from Maria Dominguez ) B . A 7d intestine in which the Intestinal Stem Cells have become Dronc homozygous mutant ( - / - ) upon expressing UAS - Flp under the regulation of Dl - Gal4 . Dl - expressing cells are labelled with UASGFP ( green ) ; there are no morphological differences between A and B . Genotype : w1118 ; ; delta - Gal4 UAS - GFP TubG80ts UAS - Histone - RFP DroncKO / UAS - Flippase ( BL8209 ) FRT Dronc - GFP - APEX FRT suntag - HA - Cherry C . Relative number of GFP + Delta - expressing cells normalised to DAPI ; there is no significant increase in intestinal stem cell number between heterozygous and homozygous Dronc mutant conditions ( ns ; P = 0 . 9231 ) ( Quantifications were made using N ≥ 2 biological replicates ; unpaired two - tailed t test , + / - n = 22 , - / - n = 13 ) . Error bars represent Standard Deviation of the Mean . Genotypes : + / - : w1118 ; ; delta - Gal4 UAS - GFP TubG80ts UAS - Histone - RFP DroncKO / UAS - Flippase ( BL8209 ) FRT Dronc - GFP - APEX FRT suntag - HA - Cherry - / - : w1118 ; ; delta - Gal4 UAS - GFP TubG80ts UAS - Histone - RFP DroncKO / UAS - Flippase ( BL8209 ) FRT Dronc - GFP - APEX FRT suntag - HA - Cherry D . Average Delta cell size ( μm² ) ; notice that the cell size does not change between heterozygous and homozygous Dronc mutant ISCs ( ns ; P = 0 . 9694 ) ( Quantifications were made using N ≥ 2 biological replicates ; unpaired two - tailed t test , + / - n = 42 , - / - n = 21 ) . Error bars represent Standard Deviation of the mean . Genotypes :

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

. A 7d intestine Dronc heterozygous (+/-) intestine in which intestinal stem cells express GFP under the regulation of Dl-Gal4. All of the experiments described in the figure were performed in Oxford medium following an experimental regime that protects the epithelial integrity (flies transferred every two days into vials with fresh food). DAPI (blue) labels the DNA in the entire Figure. Genotype: w1118; ;delta-Gal4 UAS-GFP TubG80ts UAS-Histone-RFP DroncKO/+ (recombinant chromosome made for this study; the parental line delta-Gal4 UAS-GFP TubG80ts UAS-Histone-RFP was obtained from Maria Dominguez) B. A 7d intestine in which the Intestinal Stem Cells have become Dronc homozygous mutant (-/-) upon expressing UAS-Flp under the regulation of Dl-Gal4. Dl-expressing cells are labelled with UASGFP (green); there are no morphological differences between A and B. Genotype: w1118; ;delta-Gal4 UAS-GFP TubG80ts UAS-Histone-RFP DroncKO / UAS-Flippase (BL8209) FRT Dronc-GFP-APEX FRT suntag-HA-Cherry C. Relative number of GFP+ Delta-expressing cells normalised to DAPI; there is no significant increase in intestinal stem cell number between heterozygous and homozygous Dronc mutant conditions (ns; P = 0.9231) (Quantifications were made using N ≥ 2 biological replicates; unpaired two-tailed t test, +/- n = 22, -/- n = 13). Error bars represent Standard Deviation of the Mean. Genotypes: +/- :w1118; ;delta-Gal4 UAS-GFP TubG80ts UAS-Histone-RFP DroncKO / UAS-Flippase (BL8209) FRT Dronc-GFP-APEX FRT suntag-HA-Cherry -/- : w1118; ;delta-Gal4 UAS-GFP TubG80ts UAS-Histone-RFP DroncKO / UAS-Flippase (BL8209) FRT Dronc-GFP-APEX FRT suntag-HA-Cherry D. Average Delta cell size (μm²); notice that the cell size does not change between heterozygous and homozygous Dronc mutant ISCs (ns; P = 0.9694) (Quantifications were made using N ≥ 2 biological replicates; unpaired two-tailed t test, +/- n = 42, -/- n = 21). Error bars represent Standard Deviation of the mean. Genotypes:


. A 7d intestine Dronc heterozygous ( + / - ) intestine in which intestinal stem cells express GFP under the regulation of Dl - Gal4 . All of the experiments described in the figure were performed in Oxford medium following an experimental regime that protects the epithelial integrity ( flies transferred every two days into vials with fresh food ) . DAPI ( blue ) labels the DNA in the entire Figure . Genotype : w1118 ; ; delta - Gal4 UAS - GFP TubG80ts UAS - Histone - RFP DroncKO / + ( recombinant chromosome made for this study ; the parental line delta - Gal4 UAS - GFP TubG80ts UAS - Histone - RFP was obtained from Maria Dominguez ) B . A 7d intestine in which the Intestinal Stem Cells have become Dronc homozygous mutant ( - / - ) upon expressing UAS - Flp under the regulation of Dl - Gal4 . Dl - expressing cells are labelled with UASGFP ( green ) ; there are no morphological differences between A and B . Genotype : w1118 ; ; delta - Gal4 UAS - GFP TubG80ts UAS - Histone - RFP DroncKO / UAS - Flippase ( BL8209 ) FRT Dronc - GFP - APEX FRT suntag - HA - Cherry C . Relative number of GFP + Delta - expressing cells normalised to DAPI ; there is no significant increase in intestinal stem cell number between heterozygous and homozygous Dronc mutant conditions ( ns ; P = 0 . 9231 ) ( Quantifications were made using N ≥ 2 biological replicates ; unpaired two - tailed t test , + / - n = 22 , - / - n = 13 ) . Error bars represent Standard Deviation of the Mean . Genotypes :

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

. A 7d intestine Dronc heterozygous (+/-) intestine in which intestinal stem cells express GFP under the regulation of Dl-Gal4. All of the experiments described in the figure were performed in Oxford medium following an experimental regime that protects the epithelial integrity (flies transferred every two days into vials with fresh food). DAPI (blue) labels the DNA in the entire Figure. Genotype: w1118; ;delta-Gal4 UAS-GFP TubG80ts UAS-Histone-RFP DroncKO/+ (recombinant chromosome made for this study; the parental line delta-Gal4 UAS-GFP TubG80ts UAS-Histone-RFP was obtained from Maria Dominguez) B. A 7d intestine in which the Intestinal Stem Cells have become Dronc homozygous mutant (-/-) upon expressing UAS-Flp under the regulation of Dl-Gal4. Dl-expressing cells are labelled with UASGFP (green); there are no morphological differences between A and B. Genotype: w1118; ;delta-Gal4 UAS-GFP TubG80ts UAS-Histone-RFP DroncKO / UAS-Flippase (BL8209) FRT Dronc-GFP-APEX FRT suntag-HA-Cherry C. Relative number of GFP+ Delta-expressing cells normalised to DAPI; there is no significant increase in intestinal stem cell number between heterozygous and homozygous Dronc mutant conditions (ns; P = 0.9231) (Quantifications were made using N ≥ 2 biological replicates; unpaired two-tailed t test, +/- n = 22, -/- n = 13). Error bars represent Standard Deviation of the Mean. Genotypes:


. A 7d intestine Dronc heterozygous ( + / - ) intestine in which intestinal stem cells express GFP under the regulation of Su ( H ) - Gal4 . Genotype : w1118 ; Su ( H ) - Gal4 UAS - GFP ; TubG80ts UAS - Histone - RFP DroncKO / + A 7d intestine in which the Enteroblasts have become Dronc homozygous mutant ( - / - ) upon expressing UAS - Flp under the regulation of Su ( H ) - Gal4 . Su ( H ) - Gal4 - expressing cells are labelled with UASGFP ( green ) and UAS - Histone - RFP ; note the increase in cell size when compared to E and the absence of Histone - RFP cells without GFP signal . Genotype : w1118 ; Su ( H ) - Gal4 UAS - GFP ; TubG80ts UAS - Histone - RFP DroncKO / UAS - Flippase ( BL8209 ) FRT Dronc - GFP - APEX FRT suntag - HA - Cherry Relative number of Su ( H ) - Gal4 - expressing cells normalised to DAPI ; there is no significant increase in intestinal stem cell number between heterozygous and homozygous Dronc mutant conditions ( n . s . ; P = 0 . 5099 ) ( Quantifications were made using N ≥ 2 biological replicates ; Mann - Whitney test , + / - n = 30 , - / - n = 29 ) . Error bars represent Standard Deviation of the mean . Genotypes : + / - : w1118 ; Su ( H ) - Gal4 UAS - GFP ; TubG80ts UAS - Histone - RFP DroncKO / + - / - : w1118 ; Su ( H ) - Gal4 UAS - GFP ; TubG80ts UAS - Histone - RFP DroncKO / UAS - Flippase ( BL8209 ) FRT Dronc - GFP - APEX FRT suntag - HA - Cherry H . Average Su ( H ) cell size ( μm² ) , there is a significant increase in Su ( H ) Dronc null cells ( - / - ) when compared to the heterozygous control condition ( * * ; P = 0 . 0064 ) ( Quantifications were made using N ≥ 2 biological replicates ; Mann - Whitney test , + / - n = 49 , - / - n = 33 ) . Error bars represent Standard Deviation of the mean . Genotypes :

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

. A 7d intestine Dronc heterozygous (+/-) intestine in which intestinal stem cells express GFP under the regulation of Su(H)-Gal4. Genotype: w1118; Su(H)-Gal4 UAS-GFP; TubG80ts UAS-Histone-RFP DroncKO/+ A 7d intestine in which the Enteroblasts have become Dronc homozygous mutant (-/-) upon expressing UAS-Flp under the regulation of Su(H)-Gal4. Su(H)-Gal4-expressing cells are labelled with UASGFP (green) and UAS-Histone-RFP; note the increase in cell size when compared to E and the absence of Histone-RFP cells without GFP signal. Genotype: w1118; Su(H)-Gal4 UAS-GFP; TubG80ts UAS-Histone-RFP DroncKO/ UAS-Flippase (BL8209) FRT Dronc-GFP-APEX FRT suntag-HA-Cherry Relative number of Su(H)-Gal4-expressing cells normalised to DAPI; there is no significant increase in intestinal stem cell number between heterozygous and homozygous Dronc mutant conditions (n.s. ; P = 0.5099) (Quantifications were made using N ≥ 2 biological replicates; Mann-Whitney test, +/- n = 30, -/- n = 29). Error bars represent Standard Deviation of the mean. Genotypes: +/- : w1118; Su(H)-Gal4 UAS-GFP; TubG80ts UAS-Histone-RFP DroncKO/+ -/- : w1118; Su(H)-Gal4 UAS-GFP; TubG80ts UAS-Histone-RFP DroncKO/ UAS-Flippase (BL8209) FRT Dronc-GFP-APEX FRT suntag-HA-Cherry H. Average Su(H) cell size (μm²), there is a significant increase in Su(H) Dronc null cells (-/-) when compared to the heterozygous control condition (**; P = 0.0064) (Quantifications were made using N ≥ 2 biological replicates; Mann-Whitney test, +/- n = 49, -/- n = 33). Error bars represent Standard Deviation of the mean. Genotypes:


A . Representative image of a Drosophila posterior midgut showing the expression of Armadillo ( red membranes ) , Prospero ( red nuclei of EEs ) and Dronc - TurboID ( green shows Streptavidin labelled proteins by Dronc - TurboID ) ; note the preferential enrichment of Streptavidin staining is preferentially enriched in the intestinal progenitor cells ( white arrows indicate an intestinal progenitor cell upregulating Dronc ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. Representative image of a Drosophila posterior midgut showing the expression of Armadillo (red membranes), Prospero (red nuclei of EEs) and Dronc-TurboID (green shows Streptavidin labelled proteins by Dronc-TurboID); note the preferential enrichment of Streptavidin staining is preferentially enriched in the intestinal progenitor cells (white arrows indicate an intestinal progenitor cell upregulating Dronc).


Representative image of a Drosophila posterior midgut showing the expression of Armadillo ( red membranes ) and Actin - Dronc - GFP - Myc ( green , anti - GFP ) ; note the preferential enrichment of Dronc expression in the small groups of cells formed by intestinal progenitor cells ( arrow ) . High magnification of the picture shown in B . The white arrow indicates a presumptive ISC showing low levels of Dronc compared to the flanking GFP + EBs . The arrow indicates a presumptive small ISC expressing high levels of Arm and low levels of Dronc - GFP - Myc . Transversal Z - section of the cells shown in C . The arrow indicates a presumptive small ISC expressing high levels of Arm and low levels of Dronc - GFP - Myc .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative image of a Drosophila posterior midgut showing the expression of Armadillo (red membranes) and Actin-Dronc-GFP-Myc (green, anti-GFP); note the preferential enrichment of Dronc expression in the small groups of cells formed by intestinal progenitor cells (arrow). High magnification of the picture shown in B. The white arrow indicates a presumptive ISC showing low levels of Dronc compared to the flanking GFP+ EBs. The arrow indicates a presumptive small ISC expressing high levels of Arm and low levels of Dronc-GFP-Myc. Transversal Z-section of the cells shown in C. The arrow indicates a presumptive small ISC expressing high levels of Arm and low levels of Dronc-GFP-Myc.


E . The enteroblast marker Su ( H ) ( red , immunostaining against Beta - galactosidase ) strongly co - localises with high levels of Dronc expression ( green , immunostaining against GFP ) ; white arrow indicates the enlarged area depicted in the insets .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. The enteroblast marker Su(H) (red, immunostaining against Beta-galactosidase) strongly co-localises with high levels of Dronc expression (green, immunostaining against GFP); white arrow indicates the enlarged area depicted in the insets.


F . There is extensive overlap between the expression of the EB marker Su ( H ) ( green , immunostaining against Beta - galactosidase ; white arrow ) and the apical caspase reporter DBS - S - QF ( red , immunostaining against HA ) , but it is also present in progenitor cells without Su ( H ) expression ( yellow arrowheads ) . DAPI ( Blue ) labels cell nuclei in all the panels .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F. There is extensive overlap between the expression of the EB marker Su(H) (green, immunostaining against Beta-galactosidase; white arrow) and the apical caspase reporter DBS-S-QF (red, immunostaining against HA), but it is also present in progenitor cells without Su(H) expression (yellow arrowheads). DAPI (Blue) labels cell nuclei in all the panels.


A . Representative ReDDM labeling of a Drosophila Dronc heterozygous ( + / - ) intestine overexpressing an RNAi against Notch for 3 days ; note the lack of fully differentiated Histone - RFP cells as EC ( red ) without esg expression ( green , GFP ) . DAPI staining labels cell nuclei in panels All of the experiments described in the figure were performed in Oxford medium following an experimental regime that protects the epithelial integrity . Genotype : w1118 UAS - Notch - RNAi ( Joaquin Navascués ) ; esg - Gal4 UAS - CD8 - GFP / + ; TubG80ts UAS - Histone - RFP DroncKO / + B . Representative ReDDM labelling of a Drosophila Dronc mutant homozygous ( - / - ) intestine overexpressing an RNAi against Notch for 3 post temperature shift at 29 oC ; note the lack of differentiated Histone - RFP cells as EC ( red ) without esg expression ( green , GFP ) , as well as the increase of GFP - positive cells ( compare A to B ) . Genotype : w1118 UAS - Notch - RNAi ( Joaquin Navascués ) ; esg - Gal4 UAS - CD8 - GFP / + ; TubG80ts UAS - Histone - RFP DroncKO / UAS - Flippase ( BL8209 ) FRT Dronc - GFP - APEX FRT suntag - HA - Cherry C . Quantification of the number of Histone - RFP cells normalised to DAPI ( proxy of progenitor cell proliferation obtained from the experiments shown in A and B panels ) ; note the statically significant increase in Histone - RFP positive cells in Dronc homozygous mutant intestines compared to controls ( * * ; P = 0 . 0080 ) ( Quantifications were made using N ≥ 2 biological replicates ; unpaired two - tailed t test , + / - n = 8 , - / - n = 15 ) . Error bars represent Standard Deviation of the mean . Genotypes : + / - : w1118 UAS - Notch - RNAi ; esg - Gal4 UAS - CD8 - GFP / + ; TubG80ts UAS - Histone - RFP DroncKO / + - / - : w1118 UAS - Notch - RNAi ( Joaquin Navascués ) ; esg - Gal4 UAS - CD8 - GFP / + ; TubG80ts UAS - Histone - RFP DroncKO / UAS - Flippase ( BL8209 ) FRT Dronc - GFP - APEX FRT suntag - HA - Cherry

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. Representative ReDDM labeling of a Drosophila Dronc heterozygous (+/-) intestine overexpressing an RNAi against Notch for 3 days; note the lack of fully differentiated Histone-RFP cells as EC (red) without esg expression (green, GFP). DAPI staining labels cell nuclei in panels All of the experiments described in the figure were performed in Oxford medium following an experimental regime that protects the epithelial integrity. Genotype: w1118 UAS-Notch-RNAi (Joaquin Navascués); esg-Gal4 UAS-CD8-GFP / + ; TubG80ts UAS-Histone-RFP DroncKO / + B. Representative ReDDM labelling of a Drosophila Dronc mutant homozygous (-/-) intestine overexpressing an RNAi against Notch for 3 post temperature shift at 29 oC; note the lack of differentiated Histone-RFP cells as EC (red) without esg expression (green, GFP), as well as the increase of GFP-positive cells (compare A to B). Genotype: w1118 UAS-Notch-RNAi (Joaquin Navascués); esg-Gal4 UAS-CD8-GFP / + ; TubG80ts UAS-Histone-RFP DroncKO / UAS-Flippase (BL8209) FRT Dronc-GFP-APEX FRT suntag-HA-Cherry C. Quantification of the number of Histone-RFP cells normalised to DAPI (proxy of progenitor cell proliferation obtained from the experiments shown in A and B panels); note the statically significant increase in Histone-RFP positive cells in Dronc homozygous mutant intestines compared to controls (**; P =0.0080) (Quantifications were made using N ≥ 2 biological replicates; unpaired two-tailed t test, +/- n = 8, -/- n = 15). Error bars represent Standard Deviation of the mean. Genotypes: +/-: w1118 UAS-Notch-RNAi; esg-Gal4 UAS-CD8-GFP / + ; TubG80ts UAS-Histone-RFP DroncKO / + -/-: w1118 UAS-Notch-RNAi (Joaquin Navascués); esg-Gal4 UAS-CD8-GFP / + ; TubG80ts UAS-Histone-RFP DroncKO / UAS-Flippase (BL8209) FRT Dronc-GFP-APEX FRT suntag-HA-Cherry


D . Drosophila Dronc heterozygous intestine overexpressing the Notch intracellular domain for 7 days post temperature shift at 29 oC ; intestinal esg - positive progenitor cells ( green ( GFP ) and red ( Histone - RFP ) . Genotype : w1118 ; esg - Gal4 UAS - CD8 - GFP / UAS - Notchintra ( Joaquin Navascués ) ; TubG80ts UAS - Histone - RFP DroncKO / + E . Drosophila Dronc homozygous intestine overexpressing the Notch intracellular domain for 7d post temperature shift at 29 oC ; notice that the Dronc deficiency accelerates the elimination of intestinal progenitor cells induced by Notch overactivation ( compare D and E ) . The white arrows indicate the position of insets 500μm from the posterior region . Note the complete loss of esg labelled cells in this region . Genotype : w1118 ; esg - Gal4 UAS - CD8 - GFP / UAS - Notchintra ; TubG80ts UAS - Histone - RFP DroncKO / UAS - Flippase ( BL8209 ) FRT Dronc - GFP - APEX FRT suntag - HA - Cherry F . Relative number of esg - positive cells to DAPI in either heterozygous or homozygous Dronc mutant esg cells overexpressing Notch - Intra ; note the significant reduction of esg - expressing cells ( * * * * ; P < 0 . 0001 ) ( Quantifications were made using N ≥ 2 biological replicates ; Mann - Whitney test , + / - n = 17 , - / - n = 17 ) . Error bars represent Standard Deviation of the mean . Genotypes : + / - : w1118 ; esg - Gal4 UAS - CD8 - GFP / UAS - Notchintra ; TubG80ts UAS - Histone - RFP DroncKO / + - / - : w1118 ; esg - Gal4 UAS - CD8 - GFP / UAS - Notchintra ; TubG80ts UAS - Histone - RFP DroncKO / UAS - Flippase ( BL8209 ) FRT Dronc - GFP - APEX FRT suntag - HA -

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. Drosophila Dronc heterozygous intestine overexpressing the Notch intracellular domain for 7 days post temperature shift at 29 oC; intestinal esg-positive progenitor cells (green (GFP) and red (Histone-RFP). Genotype: w1118; esg-Gal4 UAS-CD8-GFP / UAS-Notchintra (Joaquin Navascués); TubG80ts UAS-Histone-RFP DroncKO / + E. Drosophila Dronc homozygous intestine overexpressing the Notch intracellular domain for 7d post temperature shift at 29 oC; notice that the Dronc deficiency accelerates the elimination of intestinal progenitor cells induced by Notch overactivation (compare D and E). The white arrows indicate the position of insets 500μm from the posterior region. Note the complete loss of esg labelled cells in this region. Genotype: w1118; esg-Gal4 UAS-CD8-GFP / UAS-Notchintra; TubG80ts UAS-Histone-RFP DroncKO / UAS-Flippase (BL8209) FRT Dronc-GFP-APEX FRT suntag-HA-Cherry F. Relative number of esg-positive cells to DAPI in either heterozygous or homozygous Dronc mutant esg cells overexpressing Notch-Intra; note the significant reduction of esg-expressing cells (****; P < 0.0001) (Quantifications were made using N ≥ 2 biological replicates; Mann-Whitney test, +/- n = 17, -/- n = 17). Error bars represent Standard Deviation of the mean. Genotypes: +/-: w1118; esg-Gal4 UAS-CD8-GFP / UAS-Notchintra ; TubG80ts UAS-Histone-RFP DroncKO / + -/-: w1118; esg-Gal4 UAS-CD8 -GFP / UAS-Notchintra; TubG80ts UAS-Histone-RFP DroncKO / UAS-Flippase (BL8209) FRT Dronc-GFP-APEX FRT suntag-HA-Cherry


D . Drosophila Dronc heterozygous intestine overexpressing the Notch intracellular domain for 7 days post temperature shift at 29 oC ; intestinal esg - positive progenitor cells ( green ( GFP ) and red ( Histone - RFP ) . Genotype : w1118 ; esg - Gal4 UAS - CD8 - GFP / UAS - Notchintra ( Joaquin Navascués ) ; TubG80ts UAS - Histone - RFP DroncKO / +

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. Drosophila Dronc heterozygous intestine overexpressing the Notch intracellular domain for 7 days post temperature shift at 29 oC; intestinal esg-positive progenitor cells (green (GFP) and red (Histone-RFP). Genotype: w1118; esg-Gal4 UAS-CD8-GFP / UAS-Notchintra (Joaquin Navascués); TubG80ts UAS-Histone-RFP DroncKO / +


G . Representative image of a Drosophila Dronc heterozygous intestines 7 days after transfer to 29 oC . esg expression in green ( GFP ) labels all intestinal progenitors cells , whilst Su ( H ) - lacZ ( red ) distinguishes a subpopulation of EBs within the esg - expressing cells . Genotype : w1118 , Su ( H ) GBE - LacZ ; esg - Gal4 UAS - CD8 - GFP / + ; TubG80ts UAS - Histone - RFP DroncKO / +

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G. Representative image of a Drosophila Dronc heterozygous intestines 7 days after transfer to 29 oC. esg expression in green (GFP) labels all intestinal progenitors cells, whilst Su(H)-lacZ (red) distinguishes a subpopulation of EBs within the esg-expressing cells. Genotype: w1118, Su(H)GBE-LacZ; esg-Gal4 UAS-CD8-GFP / + ; TubG80ts UAS-Histone-RFP DroncKO / +


H . Representative image of a Drosophila Dronc KO intestine 7 days after transfer to 29 oC . Note , the transcription of Su ( H ) in large cells GFP ( - ) , suggestive of an aberrant upregulation of Notch - signalling within fully differentiated ECs . Genotype : w1118 , Su ( H ) GBE - LacZ ; esg - Gal4 UAS - CD8 - GFP / + ; TubG80ts UAS - Histone - RFP DroncKO / UAS - Flippase ( BL8209 ) FRT Dronc - GFP - APEX FRT suntag - HA -

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H. Representative image of a Drosophila Dronc KO intestine 7 days after transfer to 29 oC. Note, the transcription of Su(H) in large cells GFP (-), suggestive of an aberrant upregulation of Notch-signalling within fully differentiated ECs. Genotype: w1118, Su(H)GBE-LacZ; esg-Gal4 UAS-CD8-GFP / + ; TubG80ts UAS-Histone-RFP DroncKO / UAS-Flippase (BL8209) FRT Dronc-GFP-APEX FRT suntag-HA-Cherry


I . Representative image of a 7d Drosophila Dronc heterozygous intestines following a 16hr treatment with paraquat . Note the absence of large Su ( H ) positive , GFP ( - ) cells compared to ( H ) . The white arrows indicate the enlarged area depicted in the insets . Genotypes : + / - : w1118 , Su ( H ) GBE - LacZ ; esg - Gal4 UAS - CD8 - GFP / + ; TubG80ts UAS - Histone - RFP DroncKO / + - / - : w1118 , Su ( H ) GBE - LacZ ; esg - Gal4 UAS - CD8 - GFP / + ; TubG80ts UAS - Histone - RFP DroncKO / UAS - Flippase ( BL8209 ) FRT Dronc - GFP - APEX FRT suntag - HA -

I. Representative image of a 7d Drosophila Dronc heterozygous intestines following a 16hr treatment with paraquat. Note the absence of large Su(H) positive, GFP (-) cells compared to (H). The white arrows indicate the enlarged area depicted in the insets. Genotypes: +/-: w1118, Su(H)GBE-LacZ; esg-Gal4 UAS-CD8-GFP / + ; TubG80ts UAS-Histone-RFP DroncKO / + -/-: w1118, Su(H)GBE-LacZ ; esg-Gal4 UAS-CD8-GFP / + ; TubG80ts UAS-Histone-RFP DroncKO / UAS-Flippase (BL8209) FRT Dronc-GFP-APEX FRT suntag-HA-Cherry


( a - c ) At 12 - 16 h post - transfection with GFP - LC3 , NIH3T3 ( a , b ) , MEF ( b ) or HCT116 ( c ) cells containing vector or the FLIP gene were treated with Hank ' s solution for 2 - 4 h ( a ) or with 2 μM rapamycin for 1 - 3 h ( b , c ) . Subsequently , GFP - LC3 was detected using an inverted fluorescence microscope . Scale bar , 10 μm ( a ) . The number of GFP - LC3 - positive dots per cell was counted using a fluorescence microscope . Data are mean ± s . e . m . ; n = 200 - 300 cells ; three independent experiments ; * P 0 . 01 ; * * P 0 . 05 ) ( b , c ) . At 60 h post - transfection with control siRNA or cFLIP siRNA , MEF cells were transfected with GFP - LC3 ( c ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(a-c) At 12-16 h post-transfection with GFP-LC3, NIH3T3 (a, b), MEF (b) or HCT116 (c) cells containing vector or the FLIP gene were treated with Hank's solution for 2-4 h (a) or with 2 μM rapamycin for 1-3 h (b, c). Subsequently, GFP-LC3 was detected using an inverted fluorescence microscope. Scale bar, 10 μm (a). The number of GFP-LC3-positive dots per cell was counted using a fluorescence microscope. Data are mean ± s.e.m.; n = 200-300 cells; three independent experiments; *P 0.01; **P 0.05) (b, c). At 60 h post-transfection with control siRNA or cFLIP siRNA, MEF cells were transfected with GFP-LC3 (c).


( d ) Autophagosomes were visualized through scanning electron microscopy . Scale bar , 50 nm . The arrows indicate autophagosomes . Immunoblots ( IB ) of cFLIP and actin expression are shown in Supplementary Information , Fig . S1c .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(d) Autophagosomes were visualized through scanning electron microscopy. Scale bar, 50 nm. The arrows indicate autophagosomes. Immunoblots (IB) of cFLIP and actin expression are shown in Supplementary Information, Fig. S1c.


( e , f ) NIH3T3 vector and NIH3T3 - KSHV - vFLIP cells ( e ) or TREX - BCBL - Vector and TREX - BCBL - vFLIP cells ( f ) were treated with Hank ' s solution or 2 μM rapamycin for 2 - 4 h , respectively , followed by IB with anti - LC3 and anti - actin antibody .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O

(e, f) NIH3T3 vector and NIH3T3-KSHV-vFLIP cells (e) or TREX-BCBL-Vector and TREX-BCBL-vFLIP cells (f) were treated with Hank's solution or 2 μM rapamycin for 2-4 h, respectively, followed by IB with anti-LC3 and anti-actin antibody.


( g ) At 12 - 16 h post - transfection with GFP - LC3 , TREX - BCBL - vector and TREX - BCBL - vFLIP cells were treated with doxycycline for 24 h , followed by incubation with 2 μM rapamycin for an additional 12 h . GFP - LC3 was detected using an inverted fluorescence microscope . Scale bar , 5μm . The number of GFP - LC3 - positive dots per cell was counted using a fluorescence microscope . Data are mean ± s . e . m . ; n = 200 - 300 cells ; three independent experiments ; * P 0 . 01 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(g) At 12-16 h post-transfection with GFP-LC3, TREX-BCBL-vector and TREX-BCBL-vFLIP cells were treated with doxycycline for 24 h, followed by incubation with 2 μM rapamycin for an additional 12 h. GFP-LC3 was detected using an inverted fluorescence microscope. Scale bar, 5μm. The number of GFP-LC3-positive dots per cell was counted using a fluorescence microscope. Data are mean ± s.e.m.; n = 200-300 cells; three independent experiments; *P 0.01).


( h ) Autophagosomes were visualized by scanning electron microscopy . Scale bar , 50 nm .

(h) Autophagosomes were visualized by scanning electron microscopy. Scale bar, 50 nm.


( a ) BCBL cells were used for immunoprecipitation ( IP ) with mouse IgG or anti - Atg3 , followed by immunoblotting ( IB ) with anti - cFLIP antibody ( left panel ) . NIH3T3 - KSHV - vFLIP cells were used for IP with mouse IgG or anti - Flag antibody , followed by IB with anti - Atg3 antibody ( right panel ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(a) BCBL cells were used for immunoprecipitation (IP) with mouse IgG or anti-Atg3, followed by immunoblotting (IB) with anti-cFLIP antibody (left panel). NIH3T3-KSHV-vFLIP cells were used for IP with mouse IgG or anti-Flag antibody, followed by IB with anti-Atg3 antibody (right panel).


( b ) FLIP and Atg3 interaction after autophagy induction . HCT116 cells were treated with or without 2 μM rapamycin or Hank ' s solution ( HBSS ) for 4 h before IP with anti - Atg3 antibody , followed by IB with anti - cFLIP antibody .

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(b) FLIP and Atg3 interaction after autophagy induction. HCT116 cells were treated with or without 2 μM rapamycin or Hank's solution (HBSS) for 4 h before IP with anti-Atg3 antibody, followed by IB with anti-cFLIP antibody.


( c ) Schematic diagram of human Atg3 ( top ) . N , amino terminus ; FR , flexible region ; C , carboxy terminus . At 48 h post - transfection with GST or GST - Atg3 along with Flag - vFLIP ( left ) or GST or GST - Atg3 along with GFP - LC3 ( right ) , HEK293T cells were used for GST pulldown , followed by IB with the indicated antibodies .

(c) Schematic diagram of human Atg3 (top). N, amino terminus; FR, flexible region; C, carboxy terminus. At 48 h post-transfection with GST or GST-Atg3 along with Flag-vFLIP (left) or GST or GST-Atg3 along with GFP-LC3 (right), HEK293T cells were used for GST pulldown, followed by IB with the indicated antibodies.


( d ) Schematic diagram of KSHV vFLIP ( top ) . The black boxes indicate the K - α2 and K - α4 peptides . At 48 h post - transfection with V5 - Atg3 and either GST - vFLIP DED1 mutants ( middle ) or GST - vFLIP DED2 mutants ( bottom ) , HEK293T cells were used for GST pulldown , followed by IB with anti - V5 antibody .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(d) Schematic diagram of KSHV vFLIP (top). The black boxes indicate the K-α2 and K-α4 peptides. At 48 h post-transfection with V5-Atg3 and either GST-vFLIP DED1 mutants (middle) or GST-vFLIP DED2 mutants (bottom), HEK293T cells were used for GST pulldown, followed by IB with anti-V5 antibody.


( e ) At 48 h post - transfection with GST , GST - Atg3 , and either Flag - vFLIP or Flag - vFLIP mAtg3 , HEK293T cells were used for GST pulldown , followed by IB with anti - Flag antibody .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(e) At 48 h post-transfection with GST, GST-Atg3, and either Flag-vFLIP or Flag-vFLIP mAtg3, HEK293T cells were used for GST pulldown, followed by IB with anti-Flag antibody.


( f ) At 48 h post - transfection with GST - Atg3 and GFP - LC3 along with increasing amount of Flag - vFLIP , HEK293T cells were used for GST pulldown , followed by IB with anti - GFP or anti - Flag antibody .

(f) At 48 h post-transfection with GST-Atg3 and GFP-LC3 along with increasing amount of Flag-vFLIP, HEK293T cells were used for GST pulldown, followed by IB with anti-GFP or anti-Flag antibody.


( g ) NIH3T3 vector and NIH3T3 vFLIP cells were treated with 2 μM rapamycin for 4 h and used for IP with anti - Atg3 antibody , followed by IB with anti - LC3 antibody . Finally , whole - cell lysates ( WCLs ) were used for IB with the indicated antibodies to show expression ( a - g ) .

O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(g) NIH3T3 vector and NIH3T3 vFLIP cells were treated with 2 μM rapamycin for 4 h and used for IP with anti-Atg3 antibody, followed by IB with anti-LC3 antibody. Finally, whole-cell lysates (WCLs) were used for IB with the indicated antibodies to show expression (a-g).


( a ) TREX - BCBL vector cells were treated or untreated with 50 nM rapamycin for 6 days in the presence of doxycycline and subjected to scanning electron microscopy . The morphologies of more than 100 dead cells were examined to quantify the levels of apoptosis , autophagic death , apoptosis / autophagic death , and others forms of cell death .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(a) TREX-BCBL vector cells were treated or untreated with 50 nM rapamycin for 6 days in the presence of doxycycline and subjected to scanning electron microscopy. The morphologies of more than 100 dead cells were examined to quantify the levels of apoptosis, autophagic death, apoptosis/autophagic death, and others forms of cell death.


( b ) At 12 - 16 h post - transfection with GFP - LC3 , doxycycline - treated TREX - BCBL vector , TREX - BCBL - vFLIP and TREX - BCBL - vFLIP mutant cells were incubated with or without 50 nM rapamycin for 6 days ( top ) or 2 μM rapamycin for 12 h ( bottom ) . The percentage of cell death ( trypan blue staining ) and apoptosis level ( annexin V staining ) of rapamycin - treated cells was quantified as mean ± s . d . of three independent experiments . The number of GFP - LC3 - positive dots per cell was counted using a fluorescence microscope . Data are mean ± s . e . m . ; n = 200 - 300 cells ; three independent experiments ; * P < 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(b) At 12-16 h post-transfection with GFP-LC3, doxycycline-treated TREX-BCBL vector, TREX-BCBL-vFLIP and TREX-BCBL-vFLIP mutant cells were incubated with or without 50 nM rapamycin for 6 days (top) or 2 μM rapamycin for 12 h (bottom). The percentage of cell death (trypan blue staining) and apoptosis level (annexin V staining) of rapamycin-treated cells was quantified as mean ± s.d. of three independent experiments. The number of GFP-LC3-positive dots per cell was counted using a fluorescence microscope. Data are mean ± s.e.m.; n = 200-300 cells; three independent experiments; *P<0.01.


( c ) BCBL1 cells were transfected with control siRNA or Beclin1 siRNA and treated with or without rapamycin for 5 days . The percentage of cell death was determined by trypan blue staining . Data are mean ± s . e . m . ; n = 200 - 300 ; three independent experiments ; * P 0 . 05 .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(c) BCBL1 cells were transfected with control siRNA or Beclin1 siRNA and treated with or without rapamycin for 5 days. The percentage of cell death was determined by trypan blue staining. Data are mean ± s.e.m.; n = 200-300; three independent experiments; *P 0.05.


( d ) TREX - BCBL vector and TREX - BCBL - vFLIP cells were mock - treated or treated with 50 nM rapamycin for the indicated periods of time . The results were quantified as mean ± s . d . of the combined results from three independent experiments ; * P 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(d) TREX-BCBL vector and TREX-BCBL-vFLIP cells were mock-treated or treated with 50 nM rapamycin for the indicated periods of time. The results were quantified as mean ± s.d. of the combined results from three independent experiments; *P 0.001.


( e ) HEK293 cells carrying KSHV or KSHVΔvFLIP were treated with 500 nM rapamycin for 6 days . The results were quantified as mean ± s . d . of the combined results from three independent experiments ; * P 0 . 01 . Trypan blue staining and a Beckman Coulter Z2 Particle Count and size analyzer ( BC Z2 CS analyser ) were used to determine cell death and cell growth analysis ( b - e ) .

O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(e) HEK293 cells carrying KSHV or KSHVΔvFLIP were treated with 500 nM rapamycin for 6 days. The results were quantified as mean ± s.d. of the combined results from three independent experiments; *P 0.01. Trypan blue staining and a Beckman Coulter Z2 Particle Count and size analyzer (BC Z2 CS analyser) were used to determine cell death and cell growth analysis (b-e).


( b ) At 12 - 16 h post - transfection with GFP - LC3 , TREX - BCBL vector and TREX - BCBL - vFLIP cells were treated with doxycycline for 24 h , followed by incubation with 30 μM of the TAT , K - α2 or K - α4 peptide ( top left ) or the TAT , C - α2 , or C - α4 peptide ( bottom left ) for an additional 12 h . Subsequently , autophagy was quantified as means ± s . d . of the combined results from three independent experiments ( 2 μM rapamycin treatment was included as a control ) . TREX - BCBL vector and TREX - BCBL - vFLIP cells were treated with doxycycline for 24 h , followed by incubation with 0 , 30 , or 50 μM of the TAT , K - α2 , or K - α4 peptide ( top right ) or the TAT , C - α2 , or C - α4 peptide ( bottom right ) for an additional 12 h . Trypan blue staining was then used to determine cell death ( as a percentage ) . Data are mean ± s . e . m . ; n = 200 - 300 ; three independent experiments ; * P 0 . 01 ; * * P 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(b) At 12-16 h post-transfection with GFP-LC3, TREX-BCBL vector and TREX-BCBL-vFLIP cells were treated with doxycycline for 24 h, followed by incubation with 30 μM of the TAT, K-α2 or K-α4 peptide (top left) or the TAT, C-α2, or C-α4 peptide (bottom left) for an additional 12 h. Subsequently, autophagy was quantified as means ± s.d. of the combined results from three independent experiments (2 μM rapamycin treatment was included as a control). TREX-BCBL vector and TREX-BCBL-vFLIP cells were treated with doxycycline for 24 h, followed by incubation with 0, 30, or 50 μM of the TAT, K-α2, or K-α4 peptide (top right) or the TAT, C-α2, or C-α4 peptide (bottom right) for an additional 12 h. Trypan blue staining was then used to determine cell death (as a percentage). Data are mean ± s.e.m.; n = 200-300; three independent experiments; *P 0.01; **P 0.05.


( c ) At 12 - 16 h post - transfection with GFP - LC3 , TREX - BCBL vector and TREX - BCBL - vFLIP cells were treated with doxycycline for 24 h , followed by incubation with 30 μM of the TAT , K - α2 or K - α4 peptide ( top left ) or the TAT , C - α2 , or C - α4 peptide ( bottom left ) for an additional 12 h . Subsequently , autophagy was quantified as means ± s . d . of the combined results from three independent experiments ( 2 μM rapamycin treatment was included as a control ) . TREX - BCBL vector and TREX - BCBL - vFLIP cells were treated with doxycycline for 24 h , followed by incubation with 0 , 30 , or 50 μM of the TAT , K - α2 , or K - α4 peptide ( top right ) or the TAT , C - α2 , or C - α4 peptide ( bottom right ) for an additional 12 h and GFP - LC3 puncta were subsequently detected using an inverted fluorescence microscope . Scale bar , 5 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(c) At 12-16 h post-transfection with GFP-LC3, TREX-BCBL vector and TREX-BCBL-vFLIP cells were treated with doxycycline for 24 h, followed by incubation with 30 μM of the TAT, K-α2 or K-α4 peptide (top left) or the TAT, C-α2, or C-α4 peptide (bottom left) for an additional 12 h. Subsequently, autophagy was quantified as means ± s.d. of the combined results from three independent experiments (2 μM rapamycin treatment was included as a control). TREX-BCBL vector and TREX-BCBL-vFLIP cells were treated with doxycycline for 24 h, followed by incubation with 0, 30, or 50 μM of the TAT, K-α2, or K-α4 peptide (top right) or the TAT, C-α2, or C-α4 peptide (bottom right) for an additional 12 h and GFP-LC3 puncta were subsequently detected using an inverted fluorescence microscope. Scale bar, 5 μm.


( d ) KSHV - infected BCBL1 cells were treated with 30 μM of the K - α2 or the K - α4 peptide for 12 h and subjected to scanning electron microscopy . Scale bar , 50 nm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(d) KSHV-infected BCBL1 cells were treated with 30 μM of the K-α2 or the K-α4 peptide for 12 h and subjected to scanning electron microscopy. Scale bar, 50 nm.


( e ) TREX - BCBL - Vector and TREX - BCBL - vFLIP cells were treated with the K - α2 and the K - α4 ( 30 μM each ) peptide for 12 h , followed by immunoblotting ( IB ) with anti - LC3 and anti - actin antibody .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(e) TREX-BCBL-Vector and TREX-BCBL-vFLIP cells were treated with the K-α2 and the K-α4 (30 μM each) peptide for 12 h, followed by immunoblotting (IB) with anti-LC3 and anti-actin antibody.


( f ) NOD / SCID mice received an injection of 5 × 106 BCBL1 - Luc cells , followed by intraperitoneal injections with 300 μg the TAT , K - α2 or Kα - 4 peptide for three weeks ( top three panels ) . After three weeks , a group of three mice treated with the TAT peptide was challenged with 300 μg of the K - α2 or Kα - 4 peptide for an additional three weeks ( bottom panel , see Supplementary Information , Fig . S15 ) . Tumours were measured by in vivo bioluminescence imaging .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(f) NOD/SCID mice received an injection of 5 × 106 BCBL1-Luc cells, followed by intraperitoneal injections with 300 μg the TAT, K-α2 or Kα-4 peptide for three weeks (top three panels). After three weeks, a group of three mice treated with the TAT peptide was challenged with 300 μg of the K-α2 or Kα-4 peptide for an additional three weeks (bottom panel, see Supplementary Information, Fig. S15). Tumours were measured by in vivo bioluminescence imaging.


( a ) At 16 h post - transfection with GST , GST - K - α2 or GST - K - α4 along with Flag - vFLIP ( top ) or Flag - cFLIPL ( bottom ) , HEK293T cells were used for GST pulldown , followed by immunoblotting ( IB ) with anti - Flag or anti - FLIP antibody . Whole - cell lysates ( WCLs ) were used for IB with anti - GST , anti - Flag or anti - FLIP antibody .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(a) At 16 h post-transfection with GST, GST-K-α2 or GST-K-α4 along with Flag-vFLIP (top) or Flag-cFLIPL (bottom), HEK293T cells were used for GST pulldown, followed by immunoblotting (IB) with anti-Flag or anti-FLIP antibody. Whole-cell lysates (WCLs) were used for IB with anti-GST, anti-Flag or anti-FLIP antibody.


( b ) At 16 h post - transfection with V5 - Atg3 and Flag - vFLIP with increasing amounts of GST - K - α2 or GST - K - α4 , HEK293T cells were used for GST pulldown , followed by IB with anti - Flag or anti - V5 antibody or for immunoprecipitation ( IP ) with anti - V5 antibody , followed by IB with anti - Flag antibody . WCLs were used for IB with anti - GST , anti - Flag or anti - V5 antibody .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(b) At 16 h post-transfection with V5-Atg3 and Flag-vFLIP with increasing amounts of GST-K-α2 or GST-K-α4, HEK293T cells were used for GST pulldown, followed by IB with anti-Flag or anti-V5 antibody or for immunoprecipitation (IP) with anti-V5 antibody, followed by IB with anti-Flag antibody. WCLs were used for IB with anti-GST, anti-Flag or anti-V5 antibody.


( c ) At 12 - 16 h post - transfection with GST - Atg3 along with Flag - vFLIP ( first ) , Flag - cFLIPS ( second ) , or HA - LC3 ( third ) with increasing amounts of the K - α2 + K - α4 peptides ( top three panels ) or C - α2 + C - α4 peptides ( bottom panel ) , HEK293T cells were used for GST pulldown , followed by IB with anti - Flag or anti - HA antibodies . WCLs were used for IB with anti - GST and anti - Flag antibody .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(c) At 12-16 h post-transfection with GST-Atg3 along with Flag-vFLIP (first), Flag-cFLIPS (second), or HA-LC3 (third) with increasing amounts of the K-α2+K-α4 peptides (top three panels) or C-α2+C-α4 peptides (bottom panel), HEK293T cells were used for GST pulldown, followed by IB with anti-Flag or anti-HA antibodies. WCLs were used for IB with anti-GST and anti-Flag antibody.


( d ) At 12 - 16 h post - transfection with Flag - vFLIP , HA - LC3 and V5 - Atg3 , HEK293T cells were treated with increasing amounts of the K - α2 + K - α4 peptides and used for IP anti - V5 antibody , followed by IB with anti - Flag or anti - HA antibody . WCLs were used for IB with anti - V5 , anti - HA and anti - Flag antibody .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(d) At 12-16 h post-transfection with Flag-vFLIP, HA-LC3 and V5-Atg3, HEK293T cells were treated with increasing amounts of the K-α2+K-α4 peptides and used for IP anti-V5 antibody, followed by IB with anti-Flag or anti-HA antibody. WCLs were used for IB with anti-V5, anti-HA and anti-Flag antibody.


( A - J ) Confocal images of different z sections ( 0 . 2 µm of thickness ) of a neuron transfected with GFP - LC3 ( A - D ) and all sections are flattened ( E ) . Higher magnification images of the area outlined in white are shown in the inserts ( A - D ) . Different sections of images of transfected neuron with GFP - LC3 ( green ) merged with DAPI ( blue ) . Scale bar , 20 µm . ( K - O ) Three - dimensional reconstruction from serial sections using IMARIS software , cell body and process ( K ) and higher magnification of the zoomed area outlined in white ( K - I ) . Reconstructed 3D image of the same neuron using IMARIS , which evaluates AV localization in the reconstructed 3D image ( M - O ) . Enlarged areas of neuron revealed the presence of AV in soma and neurites ( N - O ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A-J) Confocal images of different z sections (0.2 µm of thickness) of a neuron transfected with GFP-LC3 (A-D) and all sections are flattened (E). Higher magnification images of the area outlined in white are shown in the inserts (A-D). Different sections of images of transfected neuron with GFP-LC3 (green) merged with DAPI (blue). Scale bar, 20 µm. (K-O) Three-dimensional reconstruction from serial sections using IMARIS software, cell body and process (K) and higher magnification of the zoomed area outlined in white (K-I). Reconstructed 3D image of the same neuron using IMARIS, which evaluates AV localization in the reconstructed 3D image (M-O). Enlarged areas of neuron revealed the presence of AV in soma and neurites (N-O).


( B ) Total AV counting results show that a significant drop in the number of AV occurs in neurons exposed to SIV - infected microglia supernatant for 3 or 24 hr . Total number of AV are significantly increased after the pretreatment with rapamycin followed by SIV - infected microglia supernatant ( Sup ) exposure for 24 hr . * * * P < 0 . 001 , * P < 0 . 05 for n = 6 experiments . All values are mean ± SEM . AV counting using 3D model reconstruction for neurons exposed to different treatments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Total AV counting results show that a significant drop in the number of AV occurs in neurons exposed to SIV-infected microglia supernatant for 3 or 24 hr. Total number of AV are significantly increased after the pretreatment with rapamycin followed by SIV-infected microglia supernatant (Sup) exposure for 24 hr. *** P<0.001, * P<0.05 for n = 6 experiments. All values are mean±SEM. AV counting using 3D model reconstruction for neurons exposed to different treatments.


( C ) Flattened images of multi - stack confocal optical slices of neurons transfected with GFP - LC3 ( green ) to label AV , and DAPI ( blue ) to label the nucleus . The five panels display a typical sample image of a neuron under control conditions as well as 3 hr and 24 hr exposure to SIV - infected microglia supernatant or pretreated with rapamycin prior the exposure to SIV - infected microglia supernatant . The right hand panels show a snap shot of a 3D outline of the neuron with AV marked as green spheres . Scale bar , 20 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Flattened images of multi-stack confocal optical slices of neurons transfected with GFP-LC3 (green) to label AV, and DAPI (blue) to label the nucleus. The five panels display a typical sample image of a neuron under control conditions as well as 3 hr and 24 hr exposure to SIV-infected microglia supernatant or pretreated with rapamycin prior the exposure to SIV-infected microglia supernatant. The right hand panels show a snap shot of a 3D outline of the neuron with AV marked as green spheres. Scale bar, 20 µm.


( A ) The number of AV in neurites decreased significantly in neurons exposed to the SIV - infected microglia supernatant for 3 or 24 hr , however ; pretreatment with rapamycin blocked this effect . * * * P < 0 . 001 , for n = 6 experiments . Scale bar , 20 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) The number of AV in neurites decreased significantly in neurons exposed to the SIV-infected microglia supernatant for 3 or 24 hr, however; pretreatment with rapamycin blocked this effect. *** P<0.001, for n = 6 experiments. Scale bar, 20 µm.


( B ) The AV number is unchanged in neuronal soma after exposure to the SIV - infected microglia supernatant for 3 or 24 hr .

O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) The AV number is unchanged in neuronal soma after exposure to the SIV-infected microglia supernatant for 3 or 24 hr.


( C and D ) Both the total length of neuronal processes ( C ) and number of brunch points ( D ) are decreased after exposure to after SIV - infected microglia supernatant treatment . * * * P < 0 . 001 , for n = 6 experiments . All values are mean ± SEM .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C and D) Both the total length of neuronal processes (C) and number of brunch points (D) are decreased after exposure to after SIV-infected microglia supernatant treatment. *** P<0.001, for n = 6 experiments. All values are mean±SEM.


( A ) The LC3 - II level is reduced after 3 or 24 hr exposure to SIV - infected microglia supernatant and this decrease effect is blocked when is pretreated with rapamycin ( 2 µM ) . The protein level of elongation complex Atg12 - Atg5 is also reduced , and again the effect is blocked in the presence of rapamycin . The protein level of p62 is increased for similar conditions with SIV - infected microglia supernatant , and the increase is blocked in the presence of rapamycin . GAPDH protein was used in these experiments as the loading control . One representative experiment of n = 4 is shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O

(A) The LC3-II level is reduced after 3 or 24 hr exposure to SIV-infected microglia supernatant and this decrease effect is blocked when is pretreated with rapamycin (2 µM). The protein level of elongation complex Atg12-Atg5 is also reduced, and again the effect is blocked in the presence of rapamycin. The protein level of p62 is increased for similar conditions with SIV-infected microglia supernatant, and the increase is blocked in the presence of rapamycin. GAPDH protein was used in these experiments as the loading control. One representative experiment of n = 4 is shown.


( B , C and D ) Ratios between LC3 - II , Atg12 - Atg5 , p62 , respectively , normalized to GAPDH . Data are reported as mean ± SEM ( n = 4 ) . * * * P < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B, C and D) Ratios between LC3-II, Atg12-Atg5, p62, respectively, normalized to GAPDH. Data are reported as mean±SEM (n = 4). *** P<0.001.


( A ) Total AV counting results show that a significant drop occurs in neurons exposed to TNF - α or NMDA for 3 or 24 hr . * * * P < 0 . 001 , * * P < 0 . 01 for n = 6 experiments . All values are mean ± SEM . ( B and C ) Distribution of AV number in soma and neurites respectively .

O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Total AV counting results show that a significant drop occurs in neurons exposed to TNF-α or NMDA for 3 or 24 hr. *** P<0.001, ** P<0.01 for n = 6 experiments. All values are mean±SEM. (B and C) Distribution of AV number in soma and neurites respectively.


( B ) The number of AV in neurites decreased significantly in neurons exposed to both TNF - α or NMDA for 3 or 24 hr . * * * P < 0 . 001 , for n = 6 experiments .

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O

(B) The number of AV in neurites decreased significantly in neurons exposed to both TNF-α or NMDA for 3 or 24 hr. *** P<0.001, for n = 6 experiments.


( C ) The AV number in soma is increased in neurons after exposure to TNF - α for 3 or 24 hr , however it remained unaffected in neurons after exposure to NMDA . * * P < 0 . 01 for n = 6 experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O

(C) The AV number in soma is increased in neurons after exposure to TNF-α for 3 or 24 hr, however it remained unaffected in neurons after exposure to NMDA. ** P<0.01 for n = 6 experiments.


( D ) Flattened images of multi - stack confocal optical slices of neurons transfected with GFP - LC3 ( green ) to label AV , and DAPI ( blue ) to label the nucleus . transfected with GFP - LC3 ( green ) to label AV , and DAPI ( blue ) to label the nucleus . The four panels display a typical sample image of a neuron under 3 hr and 24 hr exposure to TNF - α or NMDA at 25 ng / ml and 35 µM respectively . Scale bar , 20 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O

(D) Flattened images of multi-stack confocal optical slices of neurons transfected with GFP-LC3 (green) to label AV, and DAPI (blue) to label the nucleus. transfected with GFP-LC3 (green) to label AV, and DAPI (blue) to label the nucleus. The four panels display a typical sample image of a neuron under 3 hr and 24 hr exposure to TNF-α or NMDA at 25 ng/ml and 35 µM respectively. Scale bar, 20 µm.


( A ) . Neuronal survival tested by MTT . There are significant decreases of neuronal survival after exposure to SIV - infected microglia supernatant for 3 or 24 hr , which is significantly abrogated in the presence of rapamycin . Rapamycin alone has no effect . MK - 801 ( 100 µM ) also prevented the neurotoxicity induced by SIV - infected microglia supernatant . * * * P < 0 . 001 , for n = 6 experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A). Neuronal survival tested by MTT. There are significant decreases of neuronal survival after exposure to SIV-infected microglia supernatant for 3 or 24 hr, which is significantly abrogated in the presence of rapamycin. Rapamycin alone has no effect. MK-801 (100 µM) also prevented the neurotoxicity induced by SIV-infected microglia supernatant. *** P<0.001, for n = 6 experiments.


( B ) Neuronal survival was examined with MTT in cultures exposed to SIV - infected microglia supernatant for 24 hr and in the presence or absence of different drugs such as 3 - MA ( 1 mM ) or Baf ( 100 nM ) . There is no significant difference between cells exposed to SIV - infected microglia supernatant and a pretreatment with 3 - MA followed by exposure to SIV - infected microglia supernatant . However , there is a significant decrease of neuronal survival exposed to SIV - infected microglia supernatant when cells are pretreated with Baf . The SIV - infected microglia supernatant from no infected monkey ( no Sup ) doesn ' t affect neuronal survival and there is no significant difference with control samples . All values are mean ± SEM .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Neuronal survival was examined with MTT in cultures exposed to SIV-infected microglia supernatant for 24 hr and in the presence or absence of different drugs such as 3-MA (1 mM) or Baf (100 nM). There is no significant difference between cells exposed to SIV-infected microglia supernatant and a pretreatment with 3-MA followed by exposure to SIV-infected microglia supernatant. However, there is a significant decrease of neuronal survival exposed to SIV-infected microglia supernatant when cells are pretreated with Baf. The SIV-infected microglia supernatant from no infected monkey (no Sup) doesn't affect neuronal survival and there is no significant difference with control samples. All values are mean±SEM.


( A ) Relative values ( RV ) of p62 mRNA level in frontal lobe of SIVE versus SIV and uninfected ( UN ) monkeys , n = 7 for SIVE , n = 12 for SIV and n = 9 for UN , P < 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Relative values (RV) of p62 mRNA level in frontal lobe of SIVE versus SIV and uninfected (UN) monkeys, n = 7 for SIVE, n = 12 for SIV and n = 9 for UN, P<0.05.


B ) RV of p62 mRNA level in frontal cortex of HAD versus non neurological diseased subjects ( NNDS ) , n = 8 for HAD and n = 10 for NNDS , P < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B) RV of p62 mRNA level in frontal cortex of HAD versus non neurological diseased subjects (NNDS), n = 8 for HAD and n = 10 for NNDS, P<0.0001.


( C ) p62 and ubiquitin immunoreactivity on serial sections in SIVE monkeys , as well as those with HAD in brain sections from hippocampus . p62 has a diffuse immunoreactivity in UN monkeys and in human NNDS and a dot profile structures in SIVE and HAD brain tissues . Higher magnification views are shown in green insets . Black arrows indicate p62 - or ubiquitin - immunoreactive structures within the neuronal cell body or proximal processes , and red arrowheads indicate a dot - like immunoreactivity in the neuropil . Scale bar , 20 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) p62 and ubiquitin immunoreactivity on serial sections in SIVE monkeys, as well as those with HAD in brain sections from hippocampus. p62 has a diffuse immunoreactivity in UN monkeys and in human NNDS and a dot profile structures in SIVE and HAD brain tissues. Higher magnification views are shown in green insets. Black arrows indicate p62- or ubiquitin-immunoreactive structures within the neuronal cell body or proximal processes, and red arrowheads indicate a dot-like immunoreactivity in the neuropil. Scale bar, 20 µm.


Maximum projection ( 2 z - slices ) of HeLa cells immunostained for F - actin ( Phalloidin ) , Tubulin , Pericentrin and DAPI at metaphase and early anaphase showing actin accumulation around centrosomes in early anaphase . Scale bar - 10µm Quantification of F - actin ( Phalloidin ) intensity around pericentrin positive centrosomes in HeLa cells immunostained as in ( A ) , showing the increase in F - actin around centrosomes at early anaphase . Mean actin metaphase = 1 ± 0 . 03996 , n = 92 , mean actin anaphase = 1 . 875 ± 0 . 08895 , n = 121 , Student t - test , * * * * p < 0 . 0001 ;

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Maximum projection (2 z-slices) of HeLa cells immunostained for F-actin (Phalloidin), Tubulin, Pericentrin and DAPI at metaphase and early anaphase showing actin accumulation around centrosomes in early anaphase. Scale bar- 10µm Quantification of F-actin (Phalloidin) intensity around pericentrin positive centrosomes in HeLa cells immunostained as in (A), showing the increase in F-actin around centrosomes at early anaphase. Mean actin metaphase = 1 ± 0.03996, n=92, mean actin anaphase = 1.875 ± 0.08895, n=121, Student t-test, ****p<0.0001;


Time - lapse sequence from a representative Hela cell expressing Lifeact - GFP transiting from metaphase to anaphase , showing accumulation of actin in the presumptive centrosomal region , red arrows . Images represent 4z - projection . Scale bar - 10µm .

Time-lapse sequence from a representative Hela cell expressing Lifeact-GFP transiting from metaphase to anaphase, showing accumulation of actin in the presumptive centrosomal region, red arrows. Images represent 4z-projection. Scale bar-10µm.


Time - lapse sequence of a representative HeLa cell expressing GFP - alpha - tubulin and RFP - Lifeact transiting from metaphase to anaphase showing the changes in actin and microtubules . Images represent 4 z - projection of movies taken every 1min . Bottom - higher magnification view of the centrosomes C1 and C2 from time - lapse above showing a transient increase in actin around the centrosomes in early anaphase . Scale bar = 10µm , 1µm in zoom . T = 0 is one frame before anaphase onset . Dotted circle shows centrosome position .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Time-lapse sequence of a representative HeLa cell expressing GFP-alpha-tubulin and RFP-Lifeact transiting from metaphase to anaphase showing the changes in actin and microtubules. Images represent 4 z-projection of movies taken every 1min. Bottom - higher magnification view of the centrosomes C1 and C2 from time-lapse above showing a transient increase in actin around the centrosomes in early anaphase. Scale bar = 10µm, 1µm in zoom. T=0 is one frame before anaphase onset. Dotted circle shows centrosome position.


Integrated fluorescence intensity of GFP - alpha - tubulin and RFP - Lifeact around the centrosomes for a population of cells exiting mitosis , showing actin accumulation around the centrosomes and a simultaneous decrease in tubulin over time . N = 30 centrosomes ( 15 cells ) . T = 0 is one frame before anaphase onset . Graph shows mean + / - SD , values were normalized to intensity at T = 0 . Quantification and comparison of actin ( RFP - Lifeact ) and tubulin ( a - tubulin - GFP ) fluorescence intensity around the centrosomes at metaphase ( - 4 to - 1min ) , early anaphase ( 2 - 5min ) and late anaphase ( 7 - 10min ) for a population of cells . Mean actin intensity at Metaphase = 1 . 002 ± 0 . 0239 , n = 97 , Early Anaphase = 1 . 023 ± 0 . 03047 , n = 101 , Late anaphase = 0 . 9703 ± 0 . 05004 , n = 98 . Tubulin intensity at Metaphase = 1 . 008 ± 0 . 03356 , n = 106 , Early anaphase = 0 . 9748 ± 0 . 04124 , n = 116 , Late anaphase = 0 . 9276 ± 0 . 05382 , n = 101 , One - way ANOVA .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Integrated fluorescence intensity of GFP-alpha-tubulin and RFP-Lifeact around the centrosomes for a population of cells exiting mitosis, showing actin accumulation around the centrosomes and a simultaneous decrease in tubulin over time. N=30 centrosomes (15 cells). T=0 is one frame before anaphase onset. Graph shows mean +/- SD, values were normalized to intensity at T=0. Quantification and comparison of actin (RFP-Lifeact) and tubulin (a-tubulin-GFP) fluorescence intensity around the centrosomes at metaphase (-4 to -1min), early anaphase (2-5min) and late anaphase (7-10min) for a population of cells. Mean actin intensity at Metaphase =1.002 ± 0.0239, n=97, Early Anaphase = 1.023 ± 0.03047, n=101, Late anaphase = 0.9703 ± 0.05004, n=98. Tubulin intensity at Metaphase = 1.008 ± 0.03356, n=106, Early anaphase = 0.9748 ± 0.04124, n=116, Late anaphase = 0.9276 ± 0.05382, n=101, One-way ANOVA.


Stills from time - lapse of HeLa cells expressing GFP - alpha - tubulin and RFP - lifeact arrested at prometaphase with STLC ( t = 0 ) and forced to exit mitosis with Cdk1 inhibition ( Ro3306 ) imaged every 90 seconds . Scale bar = 10µm and for zoom = 4µm . n = 21 cells from 4 independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O

Stills from time-lapse of HeLa cells expressing GFP-alpha-tubulin and RFP-lifeact arrested at prometaphase with STLC (t=0) and forced to exit mitosis with Cdk1 inhibition (Ro3306) imaged every 90 seconds. Scale bar = 10µm and for zoom = 4µm. n = 21 cells from 4 independent experiments.


Quantification of actin around centrosomes when cells are forced to exit mitosis using Cdk1 inhibition as in ( A ) , showing a similar accumulation of actin as observed in bipolar divisions . Graph shows mean actin accumulation normalized to t = 0 , errors bars indicate standard deviation .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Quantification of actin around centrosomes when cells are forced to exit mitosis using Cdk1 inhibition as in (A), showing a similar accumulation of actin as observed in bipolar divisions. Graph shows mean actin accumulation normalized to t=0, errors bars indicate standard deviation.


HeLa cells expressing GFP - centrin1 arrested at prometaphase or forced mitotic exit ( Ro3306 5 ' ) and stained with phalloidin . Scale bar - 5µm and for zoom - 2µm .

O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O

HeLa cells expressing GFP-centrin1 arrested at prometaphase or forced mitotic exit (Ro3306 5') and stained with phalloidin. Scale bar- 5µm and for zoom-2µm.


The level of actin around the centrosome in ( C ) was quantified and normalised relative to metaphase and shows an increase during forced mitotic exit . STLC arrest 1 ± 0 . 01317 , n = 210 , STLC + Ro - 5mins = 1 . 251 ± 0 . 02441 , n = 144 , Unpaired t - test with Welch correction , * * * * p < 0 . 0001 . Quantification of tubulin intensity around centrosomes during prometaphase arrested cells or cells forced to exit with Cdk1 inhibitor . Tubulin intensity decreses during forced mitotic exit . STLC - DMSO - 1 ± 0 . 02407 , n = 180 , STLC - RO - 3306 - 0 . 6516 ± 0 . 02337 , n = 136 . P < 0 . 0001 , Welch ' s t test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O

The level of actin around the centrosome in (C) was quantified and normalised relative to metaphase and shows an increase during forced mitotic exit. STLC arrest 1 ± 0.01317, n=210, STLC+Ro-5mins=1.251 ± 0.02441, n=144, Unpaired t-test with Welch correction, ****p<0.0001. Quantification of tubulin intensity around centrosomes during prometaphase arrested cells or cells forced to exit with Cdk1 inhibitor. Tubulin intensity decreses during forced mitotic exit. STLC-DMSO- 1 ± 0.02407, n=180, STLC-RO-3306- 0.6516 ± 0.02337, n=136. P<0.0001, Welch's t test.


Maximum projection ( 2 - z slices ) view of Hela cells pre - treated with DMSO and 0 . 2mM CK666 for 15 mins during their mitotic exit showing that treatment with CK666 leads to reduced accumulation of actin around the centrosomes during anaphase . Scale bar - 10µm . Quantification of actin around centrosomes for cells treated with DMSO or 0 . 2mM CK666 , showing the reduction in actin accumulation around the centrosomes following CK666 treatment . DMSO - metaphase - 1 ± 0 . 4325 , n = 54 , CK666 - metaphase - 0 . 936 ± 0 . 4604 , n = 43 , DMSO - anaphase - 1 . 8 ± 0 . 9736 , n = 76 , CK666 - anaphase - 1 . 087 ± 0 . 4597 , n = 73 ; one - way ANOVA , p < 0 . 0001 . Date pooled from 3 independent experiments .

O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Maximum projection (2-z slices) view of Hela cells pre-treated with DMSO and 0.2mM CK666 for 15 mins during their mitotic exit showing that treatment with CK666 leads to reduced accumulation of actin around the centrosomes during anaphase. Scale bar-10µm. Quantification of actin around centrosomes for cells treated with DMSO or 0.2mM CK666, showing the reduction in actin accumulation around the centrosomes following CK666 treatment. DMSO-metaphase-1±0.4325, n=54, CK666-metaphase-0.936±0.4604, n=43, DMSO-anaphase-1.8±0.9736, n=76, CK666-anaphase-1.087±0.4597, n=73; one-way ANOVA, p<0.0001. Date pooled from 3 independent experiments.