( C ) Degeneration in mosaic ommatidia in ftfd clones . At this stage of degeneration approximately half of the non ‐ pigmented ( genotypically mutant ) PR cells display a degenerative phenotype , whereas virtually all pigmented ( genotypically wt ) PR cells are normal . χ2 ‐ test : 82 . 38 ; P0 . 001 . Total PR N = 310 ( pigmented ) and 327 ( not pigmented ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Degeneration in mosaic ommatidia in ftfd clones. At this stage of degeneration approximately half of the non‐pigmented (genotypically mutant) PR cells display a degenerative phenotype, whereas virtually all pigmented (genotypically wt) PR cells are normal. χ2‐test: 82.38; P0.001. Total PR N=310 (pigmented) and 327 (not pigmented).


( D ) Tangential eye sections through MARCM clone mutants for ftfd that express either no transgene or an RNAi construct against yki ( UAS - ykiIR ) with Tub - Gal4 and aged 14 days at 29 ° C . Because the transgene UAS - ykiIR is on chromosomal arm 2 L , the same as ft , clones generated with this system carry two copies of UAS - ykiIR and therefore downregulate yki very effectively . No other cell outside the clones expresses the construct and is therefore wt for yki . Mask panel is presented on the right of each section . A quantification of photoreceptor numbers ( far right ) displays a very significant increase in the number of wt ommatidia ( N = 210 and 219 from four eyes ) . * * * P0 . 001 in two ‐ tailed t ‐ test . If all classes of ommatidia are considered , χ2 ‐ test = 232 . 30 ; P0 . 001 for 3 degrees of freedom .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Tangential eye sections through MARCM clone mutants for ftfd that express either no transgene or an RNAi construct against yki (UAS-ykiIR) with Tub-Gal4 and aged 14 days at 29°C. Because the transgene UAS-ykiIR is on chromosomal arm 2 L, the same as ft, clones generated with this system carry two copies of UAS-ykiIR and therefore downregulate yki very effectively. No other cell outside the clones expresses the construct and is therefore wt for yki. Mask panel is presented on the right of each section. A quantification of photoreceptor numbers (far right) displays a very significant increase in the number of wt ommatidia (N=210 and 219 from four eyes). ***P0.001 in two‐tailed t‐test. If all classes of ommatidia are considered, χ2‐test=232.30; P0.001 for 3 degrees of freedom.


( A ) Tangential eye sections through wtsX1 mutant fly eyes . In clones for the null wtsX1 allele , degeneration inside the clones is at severe stages at 14 days .

O O O O O O O O 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) Tangential eye sections through wtsX1 mutant fly eyes. In clones for the null wtsX1 allele, degeneration inside the clones is at severe stages at 14 days.


( B ) Tangential eye sections through sav3 clones . In sav3 after enclosure many photoreceptors are intact but after 14 days at 29 ° C most sav mutant photoreceptors have degenerated .

O O O O O O O O O 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) Tangential eye sections through sav3 clones. In sav3 after enclosure many photoreceptors are intact but after 14 days at 29°C most sav mutant photoreceptors have degenerated.


( C ) Histograms showing the quantification of ommatidia with the full complement of photoreceptors in hpo , wts and sav mutant clones . The progression of loss of photoreceptors is evident and statistically significant in all cases . Mutants for hpoBF33 do not survive for 14 days at 29oC and corresponding sections are shown in Supplementary Figure 12 . N = 195 - 268 . * * * P0 . 001 , * * P0 . 01 ; * P0 . 05 in two ‐ tailed 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 O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Histograms showing the quantification of ommatidia with the full complement of photoreceptors in hpo, wts and sav mutant clones. The progression of loss of photoreceptors is evident and statistically significant in all cases. Mutants for hpoBF33 do not survive for 14 days at 29oC and corresponding sections are shown in Supplementary Figure 12. N=195-268. ***P0.001, **P0.01; *P0.05 in two‐tailed t‐test.


( A ) Regression analysis of the overgrowth versus the neurodegeneration observed in loss of function mutants ( left ) and overexpression of transgenes ( right ) . Overgrowth quantification is shown in Supplementary Figure 14 . For loss of function mutant , the neurodegeneration was quantified at 14 days in clones ( as shown in Figures 4 and 5 ) . For UAS ‐ overexpression transgenes neurodegeneration was quantified at 14 days when expressed with GMRGal4 ; UbiGal80ts . For CycD + Cdk4 , these specific data are missing and have not been plotted ; however , Supplementary Figure 13 shows that these mutants do not cause neurodegeneration in a different setup in which they are expressed during both development and adult life . In both cases the regression coefficient r2 is extremely low ( 0 . 21 and 0 . 03 ) , indicating an absence of correlation between the two processes .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Regression analysis of the overgrowth versus the neurodegeneration observed in loss of function mutants (left) and overexpression of transgenes (right). Overgrowth quantification is shown in Supplementary Figure 14. For loss of function mutant, the neurodegeneration was quantified at 14 days in clones (as shown in Figures 4 and 5). For UAS‐overexpression transgenes neurodegeneration was quantified at 14 days when expressed with GMRGal4; UbiGal80ts. For CycD+Cdk4, these specific data are missing and have not been plotted; however, Supplementary Figure 13 shows that these mutants do not cause neurodegeneration in a different setup in which they are expressed during both development and adult life. In both cases the regression coefficient r2 is extremely low (0.21 and 0.03), indicating an absence of correlation between the two processes.


( B ) Tangential eye sections through the eyes of flies expressing with GMR - Gal4 , Ubi - Gal80ts either UAS - EGFP or UAS - Yki or UAS - YkiS111A , S168A , S250A and aged 1 or 28 days . Arrows point at missing or degenerated photoreceptors . The UAS - YkiS111A , S168A , S250A is so effective that even the very low expression leaking out with this system is enough to affect development and generate mild eye roughness and overproliferation of lattice cells . Histograms showing the quantification of cell loss in eyes shown in A and also eyes from flies aged at the intermediate 14 ‐ day stage . Mild but significant degeneration is observed for both Yki forms , with respect to the negative control ; however , no statistical difference is observed between the two Yki proteins that have dramatically different effects on overgrowth . * * * P0 . 001 , * * P0 . 01 ; * P0 . 05 in two ‐ tailed 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Tangential eye sections through the eyes of flies expressing with GMR-Gal4, Ubi-Gal80ts either UAS-EGFP or UAS-Yki or UAS-YkiS111A,S168A,S250A and aged 1 or 28 days. Arrows point at missing or degenerated photoreceptors. The UAS-YkiS111A,S168A,S250A is so effective that even the very low expression leaking out with this system is enough to affect development and generate mild eye roughness and overproliferation of lattice cells. Histograms showing the quantification of cell loss in eyes shown in A and also eyes from flies aged at the intermediate 14‐day stage. Mild but significant degeneration is observed for both Yki forms, with respect to the negative control; however, no statistical difference is observed between the two Yki proteins that have dramatically different effects on overgrowth. ***P0.001, **P0.01; *P0.05 in two‐tailed t‐test.


Neurodegeneration by polyQ Atro partially requires dachs . Tangential eye sections through dGC13 clones ( marked by the absence of yellow pigment ) either in control flies or those expressing Atro75QN with the Rhodopsin1 driver and aged 28 days . Mask panels below each section exemplify clonal boundaries and PR no . for the ommatidia . Early signs of degeneration indicated by reduced complement of photoreceptors are detected specifically in the pigmented area ( arrowhead ) . At this stage , inside d mutant clones all ommatidia are unaffected and display a wt arrangement ( arrows ) . ( B ) Histograms showing the quantification of cell loss in eyes shown in ( A ) . A statistically significant rescue is obtained both for the number of wt ommatidia ( * P0 . 05 in two ‐ tailed t ‐ test ) and for all other categories of ommatidia ( χ2 = 44 . 76 ; P0 . 001 for 2 degrees of freedom ) .

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

Neurodegeneration by polyQ Atro partially requires dachs. Tangential eye sections through dGC13 clones (marked by the absence of yellow pigment) either in control flies or those expressing Atro75QN with the Rhodopsin1 driver and aged 28 days. Mask panels below each section exemplify clonal boundaries and PR no. for the ommatidia. Early signs of degeneration indicated by reduced complement of photoreceptors are detected specifically in the pigmented area (arrowhead). At this stage, inside d mutant clones all ommatidia are unaffected and display a wt arrangement (arrows). (B) Histograms showing the quantification of cell loss in eyes shown in (A). A statistically significant rescue is obtained both for the number of wt ommatidia (*P0.05 in two‐tailed t‐test) and for all other categories of ommatidia (χ2=44.76; P0.001 for 2 degrees of freedom).


( A ) EM scan of a wt ( left ) and ftfd mutant ommatidium ( right ) of a ftfd clone after 14 days . Scale bar : 1 μm . High ‐ magnification panels ( far right , top to bottom ) display autophagosomes with undigested debris , damaged mitochondria and forming phagophores ( arrowheads ) , found in ftfd mutant cells . Scale bar : 0 . 2 μm for zoom ‐ in 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

(A) EM scan of a wt (left) and ftfd mutant ommatidium (right) of a ftfd clone after 14 days. Scale bar: 1 μm. High‐magnification panels (far right, top to bottom) display autophagosomes with undigested debris, damaged mitochondria and forming phagophores (arrowheads), found in ftfd mutant cells. Scale bar: 0.2 μm for zoom‐in panels.


( B ) Graphs of the quantification of autophagic vesicles ( AV ) per photoreceptor found in EM sections of 7 ‐ day ( left ) ‐ and 14 ‐ day ( right ) ‐ old ftfd clones . Significant accumulation of AV is found in mutant cells ( not pigmented ) with respect to genotypically wt ( pigmented ) cells . * * P0 . 01 and * * * P0 . 001 in one ‐ tailed t ‐ test . N = 12 pigmented cells versus 20 non ‐ pigmented cells for the 7 ‐ day graph and 13 pigmented cells versus 21 non ‐ pigmented cells for the 14 ‐ day graph .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Graphs of the quantification of autophagic vesicles (AV) per photoreceptor found in EM sections of 7‐day (left)‐ and 14‐day (right)‐old ftfd clones. Significant accumulation of AV is found in mutant cells (not pigmented) with respect to genotypically wt (pigmented) cells. **P0.01 and ***P0.001 in one‐tailed t‐test. N=12 pigmented cells versus 20 non‐pigmented cells for the 7‐day graph and 13 pigmented cells versus 21 non‐pigmented cells for the 14‐day graph.


( C ) Confocal pictures of whole ‐ mount retinae of a ftfd clone aged 7 days and expressing GFP ∷ Atg8a ubiquitously with Tub - Gal4 . Red is phalloidin marking rhabdomeres , green is GFP and the clone is marked by the absence of β ‐ gal staining ( blue ) . Small GFP ∷ Atg8a dots accumulate specifically inside ft mutant cells ( arrow ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Confocal pictures of whole‐mount retinae of a ftfd clone aged 7 days and expressing GFP∷Atg8a ubiquitously with Tub-Gal4. Red is phalloidin marking rhabdomeres, green is GFP and the clone is marked by the absence of β‐gal staining (blue). Small GFP∷Atg8a dots accumulate specifically inside ft mutant cells (arrow).


( D ) Confocal pictures of whole ‐ mount retinae of a ftfd clone aged 7 days ( left ) and 14 days ( right ) . Red is phalloidin marking rhabdomeres , blue is p62 and the clone is marked by the absence of GFP staining ( green ) . p62 starts to gather in small dots specifically inside ft mutant cells ( arrow ) and then accumulates massively ( arrow ) in the ft mutant clones as many cells degenerate .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Confocal pictures of whole‐mount retinae of a ftfd clone aged 7 days (left) and 14 days (right). Red is phalloidin marking rhabdomeres, blue is p62 and the clone is marked by the absence of GFP staining (green). p62 starts to gather in small dots specifically inside ft mutant cells (arrow) and then accumulates massively (arrow) in the ft mutant clones as many cells degenerate.


( A ) EM scan of a 7 ‐ day ‐ old sav3 mutant ommatidium and zoom ‐ in on autophagosomes containing undigested debris ( arrowhead ) . Scale bar : 1 μm for panel on the left and 0 . 2 μm for zoom ‐ in panel on the right .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) EM scan of a 7‐day‐old sav3 mutant ommatidium and zoom‐in on autophagosomes containing undigested debris (arrowhead). Scale bar: 1 μm for panel on the left and 0.2 μm for zoom‐in panel on the right.


( B ) Graph of the quantification of autophagic vesicles ( AV ) per photoreceptor found in EM sections of 1 ‐ week ‐ old sav3 clones . Significant accumulation of AV is found in mutant cells . * * * P0 . 001 in one ‐ tailed t ‐ test . N = 45 pigmented cells versus 42 non ‐ pigmented cells .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Graph of the quantification of autophagic vesicles (AV) per photoreceptor found in EM sections of 1‐week‐old sav3 clones. Significant accumulation of AV is found in mutant cells. ***P0.001 in one‐tailed t‐test. N=45 pigmented cells versus 42 non‐pigmented cells.


( a ) Manually segmented rosettes on 3D reconstructions from Z - stacks of 25 μm of an E5 . 75 embryo imaged using lightsheet microscopy . AVE cells are in green , and all cell membranes are labelled in grey . Left : anterior view ; Middle : lateral view ( anterior to the left ) ; Right : posterior view . Scale bars : 20 μm . ( a ' ) Mouse line strategy used to perform live imaging . ( a " ) Left : Number of rosettes normalized by the total number of cells in the epiblast region in focus expressed in percentage . Right : Frequency of rosettes , normalized by the area in mm2 of the epiblast region in focus and the time of observation in minutes . Data information : Mean ± SEM , n = 41 frames from 3 embryos . ( b , c ) Rosettes outlines on optical slices from E5 . 75 ( b ) and E6 . 25 ( c ) embryos imaged by confocal microscopy . AVE cells are in green , and all cell membranes are labelled in grey . Left : anterior view ; Middle : lateral view ( anterior to the left ) ; Right : posterior view . Scale bars : 20 μm . ( b ' ) Mouse lines strategy used to perform live imaging . ( b ' ' , c ' ) Left : Number of rosettes normalized by the total number of cells expressed in percentage . Right : Density of rosettes , normalized by the area in mm2 of the epiblast region in focus . The posterior region includes the PS region , as it could not be precisely discriminated . Data information : Mean ± SEM . E5 . 75 : n anterior = 19 frames from 3 embryos , n lateral = 23 frames from 5 embryos , n posterior = 20 frames from 3 embryos . E6 . 25 : n anterior = 16 frames from 2 embryos , n lateral = 12 frames from 1 embryo and n posterior = 16 frames from 2 embryos .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Manually segmented rosettes on 3D reconstructions from Z-stacks of 25 μm of an E5.75 embryo imaged using lightsheet microscopy. AVE cells are in green, and all cell membranes are labelled in grey. Left: anterior view; Middle: lateral view (anterior to the left); Right: posterior view. Scale bars: 20 μm. (a') Mouse line strategy used to perform live imaging. (a") Left: Number of rosettes normalized by the total number of cells in the epiblast region in focus expressed in percentage. Right: Frequency of rosettes, normalized by the area in mm2 of the epiblast region in focus and the time of observation in minutes. Data information: Mean ± SEM, n=41 frames from 3 embryos. (b, c) Rosettes outlines on optical slices from E5.75 (b) and E6.25 (c) embryos imaged by confocal microscopy. AVE cells are in green, and all cell membranes are labelled in grey. Left: anterior view; Middle: lateral view (anterior to the left); Right: posterior view. Scale bars: 20 μm. (b') Mouse lines strategy used to perform live imaging. (b'', c') Left: Number of rosettes normalized by the total number of cells expressed in percentage. Right: Density of rosettes, normalized by the area in mm2 of the epiblast region in focus. The posterior region includes the PS region, as it could not be precisely discriminated. Data information: Mean ± SEM. E5.75: n anterior=19 frames from 3 embryos, n lateral=23 frames from 5 embryos, n posterior=20 frames from 3 embryos. E6.25: n anterior=16 frames from 2 embryos, n lateral=12 frames from 1 embryo and n posterior=16 frames from 2 embryos.


( a ) Z - projection of an E7 . 25 embryo mosaically labelled through OH - tamoxifen injection , imaged by two - photon microscopy . Scale bar : 50 μm . Data information : n embryos = 54 . ( a ' ) Mouse line strategy used to produce embryos ubiquitous for membrane tdTomato ( grey ) and mosaic for membrane GFP ( mGFP , green ) in the epiblast . ( b ) Z - projections of stills from live imaging recording of a bottle - shaped cell ( indicated by a white arrow ) that delaminates through the basal membrane at time 0min , 25 min , 50 min , 1h15min and 1h40min . ( b ' ) Top : Bottle shape events : GFP + bottle shape cells over total GFP + cells , expressed in percentage , in anterior versus posterior region of the epiblast . The posterior region includes the PS region , as it could not be precisely discriminated . Percentage of bottle shape cells exiting the epiblast among total number of GFP + bottle shape cells , in anterior versus posterior epiblast . white dotted lines outline basal ( top ) and apical ( bottom ) borders of the epiblast .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Z-projection of an E7.25 embryo mosaically labelled through OH-tamoxifen injection, imaged by two-photon microscopy. Scale bar: 50 μm. Data information: n embryos=54. (a') Mouse line strategy used to produce embryos ubiquitous for membrane tdTomato (grey) and mosaic for membrane GFP (mGFP, green) in the epiblast. (b) Z-projections of stills from live imaging recording of a bottle-shaped cell (indicated by a white arrow) that delaminates through the basal membrane at time 0min, 25 min, 50 min, 1h15min and 1h40min. (b') Top: Bottle shape events: GFP+ bottle shape cells over total GFP+ cells, expressed in percentage, in anterior versus posterior region of the epiblast. The posterior region includes the PS region, as it could not be precisely discriminated. Percentage of bottle shape cells exiting the epiblast among total number of GFP+ bottle shape cells, in anterior versus posterior epiblast. white dotted lines outline basal (top) and apical (bottom) borders of the epiblast.


( c ) Z projection ( c ) and 3D reconstruction ( c ' ) of stills from live imaging recording showing an apical division without basal contact , followed by daughter cells dispersion at 0min , 25 min , 50 min , 1h15min and 1h40min . white dotted lines outline basal ( top ) and apical ( bottom ) borders of the epiblast . ( c " ) Left : Frequency of apical division ( GFP + apical division over total GFP + cells , expressed in percentage ) in anterior and posterior epiblast . Middle : Apical division with or without persistence of contact with the epiblast basal pole , normalized by the total number of GFP + apical division , in anterior versus posterior epiblast . Right : Cell dispersion ( defined as loss of basolateral contact between daughter cells after reinsertion in the epiblast layer ) after apical division in anterior and posterior epiblast .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Z projection (c) and 3D reconstruction (c') of stills from live imaging recording showing an apical division without basal contact, followed by daughter cells dispersion at 0min, 25 min, 50 min, 1h15min and 1h40min. white dotted lines outline basal (top) and apical (bottom) borders of the epiblast. (c") Left: Frequency of apical division (GFP+ apical division over total GFP+ cells, expressed in percentage) in anterior and posterior epiblast. Middle: Apical division with or without persistence of contact with the epiblast basal pole, normalized by the total number of GFP+ apical division, in anterior versus posterior epiblast. Right: Cell dispersion (defined as loss of basolateral contact between daughter cells after reinsertion in the epiblast layer) after apical division in anterior and posterior epiblast.


( a ) Z - projection ( a ) and 3D cell reconstruction ( a ' ) of stills from two - photon live imaging of an E7 . 25 embryo mosaically labelled for epiblast cells showing a division giving rise to an epiblast and a mesoderm cell that will exit from the epiblast layer . White dotted lines outline basal ( top ) and apical ( bottom ) borders of the epiblast at 0min , 25 min , 50min , 1h15min and 1h40min . ( b ) ( c ) Z projection of stills from two - photon live imaging of an E7 . 25 embryo mosaically labelled for epiblast cells . Non - apical division can be observed in the posterior epiblast only , without ( b ) or with ( c ) attachment to the apical side . Stills were taken at 0min , 25 min , 50min , 1h15min and 1h40min for ( b ) and 50min , 1h15min , 1h40min , 2h05min and 2h30min for ( c ) . White dotted lines outline basal ( top ) and apical ( bottom ) borders of the epiblast .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Z-projection (a) and 3D cell reconstruction (a') of stills from two-photon live imaging of an E7.25 embryo mosaically labelled for epiblast cells showing a division giving rise to an epiblast and a mesoderm cell that will exit from the epiblast layer. White dotted lines outline basal (top) and apical (bottom) borders of the epiblast at 0min, 25 min, 50min, 1h15min and 1h40min. (b) (c) Z projection of stills from two-photon live imaging of an E7.25 embryo mosaically labelled for epiblast cells. Non-apical division can be observed in the posterior epiblast only, without (b) or with (c) attachment to the apical side. Stills were taken at 0min, 25 min, 50min, 1h15min and 1h40min for (b) and 50min, 1h15min, 1h40min, 2h05min and 2h30min for (c). White dotted lines outline basal (top) and apical (bottom) borders of the epiblast.


( d ) Mitotic Index ( GFP + dividing cells over total number of GFP + cells ) in the anterior and posterior region , as well as among cells exiting the epiblast at the PS . The posterior region includes the PS region , as it could not be precisely discriminated .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Mitotic Index (GFP+ dividing cells over total number of GFP+ cells) in the anterior and posterior region, as well as among cells exiting the epiblast at the PS. The posterior region includes the PS region, as it could not be precisely discriminated.


( e ) Ratio of non - apical rounding ( non - apical GFP + rounding divided by the total GFP + rounding ) and expressed in percentage for the anterior and posterior regions .

O O O O O O O O O O O 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) Ratio of non-apical rounding (non-apical GFP+ rounding divided by the total GFP+ rounding) and expressed in percentage for the anterior and posterior regions.


( f ) Ratio of non - apical division ( non - apical GFP + division over total GFP + division ) and expressed in percentage for the anterior and posterior region of the embryo .

O O O O O O O O O O O O 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) Ratio of non-apical division (non-apical GFP+ division over total GFP+ division) and expressed in percentage for the anterior and posterior region of the embryo.


( g ) Quantification of posterior cell division outcome for apical and non - apical divisions . Outcomes are 2 epiblast daughter cells ( epi / epi ) , one epiblast and one mesoderm daughter cells ( epi / meso ) or 2 mesoderm daughter cells ( meso / meso ) .

(g) Quantification of posterior cell division outcome for apical and non-apical divisions. Outcomes are 2 epiblast daughter cells (epi/epi), one epiblast and one mesoderm daughter cells (epi/meso) or 2 mesoderm daughter cells (meso/meso).


( c ) Z - projections of transverse sections from E6 . 5 ( top ) , E6 . 75 ( mid ) and E7 ( bottom ) embryos . Samples were stained for F - actin ( Phalloidin , grey ) and mitosis ( Phh3 , magenta ) on the left and nuclei ( DAPI , cyan ) and basement membrane ( collagen IV , yellow ) on the right . Yellow arrowheads show non - apical mitosis . White arrows delimitate borders of the basement membrane degradation region . A : anterior , P : posterior .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Z-projections of transverse sections from E6.5 (top), E6.75 (mid) and E7 (bottom) embryos. Samples were stained for F-actin (Phalloidin, grey) and mitosis (Phh3, magenta) on the left and nuclei (DAPI, cyan) and basement membrane (collagen IV, yellow) on the right. Yellow arrowheads show non-apical mitosis. White arrows delimitate borders of the basement membrane degradation region. A: anterior, P: posterior.


( d ) Mitotic Index ( Phh3 / DAPI ) in percentage at the anterior , posterior , and PS regions of E6 . 5 ( top ) , E6 . 75 ( mid ) and E7 ( bottom ) embryos . The posterior region quantification excludes counts from the PS region .

(d) Mitotic Index (Phh3/DAPI) in percentage at the anterior, posterior, and PS regions of E6.5 (top), E6.75 (mid) and E7 (bottom) embryos. The posterior region quantification excludes counts from the PS region.


( e ) Ratio of non - apical mitosis ( non - apical Phh3 / Phh3 , normalized by number of nuclei ) in percentage at the anterior , posterior , and PS regions of E6 . 5 ( top ) , E6 . 75 ( mid ) and E7 ( bottom ) embryos .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Ratio of non-apical mitosis (non-apical Phh3/ Phh3, normalized by number of nuclei) in percentage at the anterior, posterior, and PS regions of E6.5 (top), E6.75 (mid) and E7 (bottom) embryos.


( f ) Violin plots representing the distribution of cells in G2 / M phase in cells with high , compared to low , Brachyury expression , among cells annotated as primitive streak and mesoderm harvested from E6 . 5 , E7 , and E7 . 25 embryos . Violin plots are overlaid with white box plots marking 25th percentile , median and 75th percentile .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Violin plots representing the distribution of cells in G2/M phase in cells with high, compared to low, Brachyury expression, among cells annotated as primitive streak and mesoderm harvested from E6.5, E7, and E7.25 embryos. Violin plots are overlaid with white box plots marking 25th percentile, median and 75th percentile.


( a ) 3D reconstruction from wholemount lightsheet imaging of an E5 . 75 Hex - GFP ( green , anterior marker ) embryo , stained for nuclei ( DAPI , grey ) on the left , mitosis ( Phh3 , magenta ) and F - actin ( Phalloidin , grey ) on the right . ( b ) Z - slice ( left ) and 3D reconstruction ( right ) from wholemount lightsheet imaging of an E6 . 25 embryo stained for a posterior marker ( Eomesodermin , cyan ) , mitosis ( Phh3 , magenta ) and F - actin ( Phalloidin , grey ) . ( a ' , b ' ) Mitotic Index ( Phh3 / DAPI ) in percentage at the anterior and posterior regions of E5 . 75 ( a ' ) and ( b ' ) E6 . 25 embryos . Posterior region includes the PS region , as it could not be precisely discriminated . ( a " , b " ) Ratio of non - apical mitosis ( non - apical Phh3 over total Phh3 , normalized by number of nuclei ) in percentage in the anterior and posterior region of E5 . 75 ( a " ) and E6 . 25 ( b " ) embryos .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 3D reconstruction from wholemount lightsheet imaging of an E5.75 Hex-GFP (green, anterior marker) embryo, stained for nuclei (DAPI, grey) on the left, mitosis (Phh3, magenta) and F-actin (Phalloidin, grey) on the right. (b) Z-slice (left) and 3D reconstruction (right) from wholemount lightsheet imaging of an E6.25 embryo stained for a posterior marker (Eomesodermin, cyan), mitosis (Phh3, magenta) and F-actin (Phalloidin, grey). (a', b') Mitotic Index (Phh3/DAPI) in percentage at the anterior and posterior regions of E5.75 (a') and (b') E6.25 embryos. Posterior region includes the PS region, as it could not be precisely discriminated. (a", b") Ratio of non-apical mitosis (non-apical Phh3 over total Phh3, normalized by number of nuclei) in percentage in the anterior and posterior region of E5.75 (a") and E6.25 (b") embryos.


( b , c ) 3D reconstruction ( b ) and confocal Z - slice ( c ) of E6 . 25 WT ( left ) and RhoAVE - deleted ( right ) embryos stained for an AVE marker ( Cerberus 1 , yellow ) , F - actin ( Phalloidin , grey ) and mitosis ( Phh3 , magenta ) . Scale bar : 25 μm . Blue dotted line delimitates the pro - amniotic cavity ( apical side of the epiblast ) . Red arrowheads point to non - apical mitosis . White lines delimitates the boundary between extraembryonic ( up ) and embryonic ( down ) regions of the embryo .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 3D reconstruction (b) and confocal Z-slice (c) of E6.25 WT (left) and RhoAVE-deleted (right) embryos stained for an AVE marker (Cerberus 1, yellow), F-actin (Phalloidin, grey) and mitosis (Phh3, magenta). Scale bar: 25 μm. Blue dotted line delimitates the pro-amniotic cavity (apical side of the epiblast). Red arrowheads point to non-apical mitosis. White lines delimitates the boundary between extraembryonic (up) and embryonic (down) regions of the embryo.


( d ) Percentage of AVE migration in WT and RhoAVE - deleted mutants . Data information : Values are shown as Mean ± SEM . WT : n = 31 embryos ; RhoAVE - deleted : n = 17 embryos .

O O O O O O O O O O O O O O O O O O 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) Percentage of AVE migration in WT and RhoAVE-deleted mutants. Data information: Values are shown as Mean ± SEM. WT: n=31 embryos; RhoAVE-deleted: n=17 embryos.


( e ) Ratio of non - apical mitosis ( non - apical Phh3 over total Phh3 , normalised by the volume in μm3 ) in percentage , in AVE - adjacent and AVE - opposed regions from WT and RhoAVE - deleted embryos .

O O O O O O O O O O O O O O O O O O O O O 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) Ratio of non-apical mitosis (non-apical Phh3 over total Phh3, normalised by the volume in μm3) in percentage, in AVE-adjacent and AVE-opposed regions from WT and RhoAVE-deleted embryos.


( A ) Tamoxifen schedule : pulse of the pregnant dam at E9 . 5 , harvest at E10 . 5 . Representative fluorescent immune - staining of E10 . 5 Sox7iECKO mutants and sibling control embryos . The endothelium is delineated by endomucin ( EMCN ) ( blue ) and endothelial cells by ETS - related gene ( ERG ) ( red ) , SOX7 positive cells are shown in white . ( Top right graph ) The percentage of SOX7 positive endothelial cells in different vascular beds , quantified from 10 sequential transverse sections in 2 control and 3 Sox7iECKO mutants . ( Bottom right panel ) Graph indicating the levels of Sox7 transcript in sibling controls and Sox7iECKO mutants . Mean ± SEM ; scored sibling control , n = 9 ; Sox7iECKO mutants , n = 7 ; Mann - Whitney U - test . P < 0 . 0005 ( * * * ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Tamoxifen schedule: pulse of the pregnant dam at E9.5, harvest at E10.5. Representative fluorescent immune-staining of E10.5 Sox7iECKO mutants and sibling control embryos. The endothelium is delineated by endomucin (EMCN) (blue) and endothelial cells by ETS-related gene (ERG) (red), SOX7 positive cells are shown in white. (Top right graph) The percentage of SOX7 positive endothelial cells in different vascular beds, quantified from 10 sequential transverse sections in 2 control and 3 Sox7iECKO mutants. (Bottom right panel) Graph indicating the levels of Sox7 transcript in sibling controls and Sox7iECKO mutants. Mean ± SEM; scored sibling control, n=9; Sox7iECKO mutants, n=7; Mann-Whitney U-test. P<0.0005 (***).


( C ) Brightfield images and whole - mount immunostaining of Sox7iECKO mutant and sibling control skin at E13 . 5 , after Cre induction at E9 . 5 and E10 . 5 . Dermal lymphatic structures are marked by Neuropilin 2 ( NRP2 ) ( membranous white ) , lymphatic endothelial cells by Prospero - related homeobox 1 ( PROX1 ) ( red ) , and blood vessels by EMCN ( green ) . Dash line represents the midline of the embryo . Scored sibling controls , n = 7 ; Sox7iECKO mutants , 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

(C) Brightfield images and whole-mount immunostaining of Sox7iECKO mutant and sibling control skin at E13.5, after Cre induction at E9.5 and E10.5. Dermal lymphatic structures are marked by Neuropilin 2 (NRP2) (membranous white), lymphatic endothelial cells by Prospero-related homeobox 1 (PROX1) (red), and blood vessels by EMCN (green). Dash line represents the midline of the embryo. Scored sibling controls, n=7; Sox7iECKO mutants, n=7


( D - H ) Quantification of ( D ) Distance of the lymphatic vessel front from the midline ( μm ) ( E ) lymphatic branch points / area ( F ) lymphatic vessel width ( μm ) ( G ) PROX1 + nuclei / area and ( H ) PROX1 + nuclei / lymphatic vessels across the whole skin . Total area = 4200 x 1500 μm on both sides of the midline , in sibling controls ( n = 3 ) and Sox7iECKO mutants ( n = 5 - 6 ) . ( F , H ) Average of width and PROX1 + nuclei was obtained from 7 random representative leading lymphatic vessels , of fixed length at 200 μm , from both sides of the midline in each skin . ( I , J ) Quantification of ( I ) PROX1 + nuclei in each LEC cluster with each colour dot representing a different embryo and ( J ) disconnected lymphatic endothelial cell ( LEC ) clusters ( < 100 μm ) and vessel branches ( > 100μm ) / area . Total area quantified = 4200 x 2200 μm on both side of midline in sibling controls ( n = 3 ) and Sox7iECKO mutants ( n = 5 ) . PROX1 + nuclei were quantified from n = 29 and n = 122 LEC clusters , respectively .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-H) Quantification of (D) Distance of the lymphatic vessel front from the midline (μm) (E) lymphatic branch points/area (F) lymphatic vessel width (μm) (G) PROX1+ nuclei/area and (H) PROX1+ nuclei/lymphatic vessels across the whole skin. Total area = 4200 x 1500 μm on both sides of the midline, in sibling controls (n=3) and Sox7iECKO mutants (n=5-6). (F,H) Average of width and PROX1+ nuclei was obtained from 7 random representative leading lymphatic vessels, of fixed length at 200 μm, from both sides of the midline in each skin. (I,J) Quantification of (I) PROX1+ nuclei in each LEC cluster with each colour dot representing a different embryo and (J) disconnected lymphatic endothelial cell (LEC) clusters (<100 μm) and vessel branches (>100μm)/area. Total area quantified = 4200 x 2200 μm on both side of midline in sibling controls (n=3) and Sox7iECKO mutants (n=5). PROX1+ nuclei were quantified from n=29 and n=122 LEC clusters, respectively.


( C ) Arterial endothelial cells express Vegfc transcripts . Transverse section of a E10 . 5 wild - type embryo stained by single molecule FISH ( smFISH ) for Gapdh ( green ) and Vegfc ( magenta ) and immuno - stained for PECAM ( white ) . The first panel shows a merged version of the pseudo colour signals for the 3 markers . Inset 1 shows the expression of Vegfc in endothelial cell of the dorsal aorta ( black and white , pink arrows ) . Inset 2 shows the high expression levels of mesenchymal Vegfc expressed in the dorso - lateral aspects of the cardinal vein .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Arterial endothelial cells express Vegfc transcripts. Transverse section of a E10.5 wild-type embryo stained by single molecule FISH (smFISH) for Gapdh (green) and Vegfc (magenta) and immuno-stained for PECAM (white). The first panel shows a merged version of the pseudo colour signals for the 3 markers. Inset 1 shows the expression of Vegfc in endothelial cell of the dorsal aorta (black and white, pink arrows). Inset 2 shows the high expression levels of mesenchymal Vegfc expressed in the dorso-lateral aspects of the cardinal vein.


( E - F ) qPCR on human arterial endothelial cells ( HUAECs ) transfected with SiSOX7 or SiCTRL for ( E ) 30 h and ( F ) 17 h . ( G ) qPCR on human venous endothelial cells ( HUVECS ) transfected with SiSOX7 or SiCTRL for 17 h .

(E-F) qPCR on human arterial endothelial cells (HUAECs) transfected with SiSOX7 or SiCTRL for (E) 30 h and (F) 17 h. (G) qPCR on human venous endothelial cells (HUVECS) transfected with SiSOX7 or SiCTRL for 17 h. D


( A - D ) SOX7 transcriptionally activates Notch effector , HEY1 , to repress Vegfc . ( A ) qPCR on FAC - sorted PECAM + CD45 - endothelial cells of Sox7iECKO mutants and sibling controls at E14 . 5 after Cre induction with tamoxifen at E11 . 5 and E12 . 5 . Expression is normalised to the endothelial marker Pecam and Cdh5 . Scored sibling controls , n = 9 ; Sox7iECKO mutants , n = 8 . ( B - C ) qPCR on ( B ) human arterial endothelial cells ( HUAECs ) and ( C ) human venous endothelial cells ( HUVECs ) transfected with SiSOX7 or SiCTRL for 17 h . In addition to HEY1 , DLL4 levels were also downregulated in the human cell line experiments . Expression is relative to HPRT and GAPDH . Data from one siRNA experiment performed in triplicates . ( D ) HEY1 represses human VEGFC promoter activity . HeLa cells were co - transfected with human or mouse VEGFC - luc and either EV ( empty vector ) or HEY1 expression constructs as indicated . VEGFC luciferase activity was measured and normalised to Renillla luciferase activity , which was then made relative to the promoter - less vector , pGL3 - basic , which was set to 1 . Biological replicates , n = 3 independent repeats of the same experiment . Mean ± SEM ; t - test . P < 0 . 05 ( * ) ; P < 0 . 005 ( * * ) ; P < 0 . 0005 ( * * * ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-D) SOX7 transcriptionally activates Notch effector, HEY1, to repress Vegfc. (A) qPCR on FAC-sorted PECAM+CD45-endothelial cells of Sox7iECKO mutants and sibling controls at E14.5 after Cre induction with tamoxifen at E11.5 and E12.5. Expression is normalised to the endothelial marker Pecam and Cdh5. Scored sibling controls, n=9; Sox7iECKO mutants, n=8. (B-C) qPCR on (B) human arterial endothelial cells (HUAECs) and (C) human venous endothelial cells (HUVECs) transfected with SiSOX7 or SiCTRL for 17 h. In addition to HEY1, DLL4 levels were also downregulated in the human cell line experiments. Expression is relative to HPRT and GAPDH. Data from one siRNA experiment performed in triplicates. (D) HEY1 represses human VEGFC promoter activity. HeLa cells were co-transfected with human or mouse VEGFC-luc and either EV (empty vector) or HEY1 expression constructs as indicated. VEGFC luciferase activity was measured and normalised to Renillla luciferase activity, which was then made relative to the promoter-less vector, pGL3-basic, which was set to 1. Biological replicates, n=3 independent repeats of the same experiment. Mean ± SEM; t-test. P<0.05 (*); P<0.005 (**); P<0.0005 (***).


SOX7 physically interacts with transcription repressor , HEY1 . ( G ) Co - immnuno - precipitation analysis of SOX7 and HEY1 . HEK293 cells were transfected with indicated plasmids and harvested 24 h after transfection , immunoprecipitated by anti - GFP or Ig control before immunoblot analysis to determine the presence of bait / prey ( top ) and the input ( bottom ) . N = 2 .

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

SOX7 physically interacts with transcription repressor, HEY1. (G) Co-immnuno-precipitation analysis of SOX7 and HEY1. HEK293 cells were transfected with indicated plasmids and harvested 24 h after transfection, immunoprecipitated by anti-GFP or Ig control before immunoblot analysis to determine the presence of bait/prey (top) and the input (bottom). N=2.


( A ) SOX7 represses human VEGFC promoter activity . HeLa cells were co - transfected with human or mouse VEGFC - luc , EV ( empty vector ) , or SOX7 expression constructs as indicated . VEGFC luciferase activity was measured and normalised to Renillla luciferase activity , which was then made relative to the promoter - less vector , pGL3 - basic , which was set to 1 . Biological replicates , n = 3 . Mean ± SEM ; t - test . 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

(A) SOX7 represses human VEGFC promoter activity. HeLa cells were co-transfected with human or mouse VEGFC-luc, EV (empty vector), or SOX7 expression constructs as indicated. VEGFC luciferase activity was measured and normalised to Renillla luciferase activity, which was then made relative to the promoter-less vector, pGL3-basic, which was set to 1. Biological replicates, n=3. Mean ± SEM; t-test. P<0.05 (*).


( G ) Quantitation of Vegfc transcript using single molecule FISH in vivo . ( colored panel ) Representative low magnification images of the trunk transverse sections of E10 . 5 sibling controls and Sox7iECKO mutants showing the similar relative position of the DA and the CV across genotypes . Sections were stained by single molecule FISH ( smFISH ) for Gapdh ( green ) and Vegfc ( magenta ) and immuno - stained for PECAM ( white ) . Individual molecules of Vegfc transcript ( pink arrows ) are detectable within the cytoplasm of PECAM positive cells of the dorsal aorta ( Middle panel ) . ( Left graph ) Quantitation of Vegfc transcript level in the PECAM positive endothelial cells of the dorsal aorta , in sibling controls and Sox7iECKO mutants at E10 . 5 . The mean grey scale intensity of the Vegfc probes in control was 5845 . The mean grey scale intensity of the Vegfc probes in Sox7iECKO was 8956 . Mean probe intensity was significantly higher in Sox7iECKO compared to the control ( P < 2e - 16 ) . ( Right graph ) No differences were observed for the average probe area , meaning that distribution of the probes across the dorsal aorta remained unchanged between control and Sox7iECKO . Scored sibling controls , n = 5 ; Sox7iECKO mutants , n = 7 . Mean ± SEM ; t - test . P < 0 . 0005 ( * * * ) , Graph : box , interquartile range , central band , median , whiskers , indicate maximum and minimum . Scale bars ; low magnification 150 μm , high magnification , 30

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantitation of Vegfc transcript using single molecule FISH in vivo. (colored panel) Representative low magnification images of the trunk transverse sections of E10.5 sibling controls and Sox7iECKO mutants showing the similar relative position of the DA and the CV across genotypes. Sections were stained by single molecule FISH (smFISH) for Gapdh (green) and Vegfc (magenta) and immuno-stained for PECAM (white). Individual molecules of Vegfc transcript (pink arrows) are detectable within the cytoplasm of PECAM positive cells of the dorsal aorta (Middle panel). (Left graph) Quantitation of Vegfc transcript level in the PECAM positive endothelial cells of the dorsal aorta, in sibling controls and Sox7iECKO mutants at E10.5. The mean grey scale intensity of the Vegfc probes in control was 5845. The mean grey scale intensity of the Vegfc probes in Sox7iECKO was 8956. Mean probe intensity was significantly higher in Sox7iECKO compared to the control (P<2e-16). (Right graph) No differences were observed for the average probe area, meaning that distribution of the probes across the dorsal aorta remained unchanged between control and Sox7iECKO. Scored sibling controls, n=5; Sox7iECKO mutants, n=7. Mean ± SEM; t-test. P<0.0005 (***), Graph: box, interquartile range, central band, median, whiskers, indicate maximum and minimum. Scale bars; low magnification 150 μm, high magnification, 30 μm


( A ) At 2 days post Cre induction , E11 . 5 Sox7iECKO mutants showed ectopic expression of PROX1 + ( red ) in medial ventral regions of the cardinal veins ( CVs ) , close to dorsal aorta ( DA ) ( yellow arrowheads ) .

O O O O O O O O O O O O O O O O O O O O O O O O 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 2 days post Cre induction, E11.5 Sox7iECKO mutants showed ectopic expression of PROX1+ (red) in medial ventral regions of the cardinal veins (CVs), close to dorsal aorta (DA) (yellow arrowheads).


( D ) Whole - mount immunostaining of control and Sox7iECKO embryonic skin at E12 . 5 after Cre induction at E9 . 5 , E10 . 5 . Dermal lymphatic structures are stained with NRP2 ( membranous white ) , lymphatic endothelial cells , PROX1 ( red ) , and blood vessels , both EMCN ( green ) and SOX7 ( white nuclei ) . Arrowheads indicate EMCNlow / - PROX1 + NRP2 + / low individual LECs on blood vessels ( type I ) , and arrow shows EMCN + PROX1 + NRP2low / - LEC within the vessel wall ( type II ) . ( E ) Graph shows the total events of PROX1 + single LECs or LEC clusters cells associated with EMCN + blood vessel plexus within each embryo . PROX1 + cells were quantified from n = 4 controls and n = 5 Sox7iECKO mutants .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Whole-mount immunostaining of control and Sox7iECKO embryonic skin at E12.5 after Cre induction at E9.5, E10.5. Dermal lymphatic structures are stained with NRP2 (membranous white), lymphatic endothelial cells, PROX1 (red), and blood vessels, both EMCN (green) and SOX7 (white nuclei). Arrowheads indicate EMCNlow/-PROX1+NRP2+/low individual LECs on blood vessels (type I), and arrow shows EMCN+PROX1+ NRP2low/- LEC within the vessel wall (type II). (E) Graph shows the total events of PROX1+ single LECs or LEC clusters cells associated with EMCN+ blood vessel plexus within each embryo. PROX1+ cells were quantified from n=4 controls and n=5 Sox7iECKO mutants.


Purified CUL3WT and CUL3Δ403 - 459 analysed by SDS - PAGE and Coomassie blue staining , following expression in SF21 cells , and DAC - tag - affinity purification and tag cleavage ( see Materials and Methods ) . Bands migrating between 62 - and 98 - kDa protein markers are CUL3WT and CUL3Δ403 - 459 , respectively , while the band below 17 kDa is Rbx1 . The immunoblot for RBX1 is shown on the right . The band in CUL3WT denoted with * is co - purified insect cell CAND1 , which was later removed by SEC . All protein identities were confirmed by mass spectrometry .

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

Purified CUL3WT and CUL3Δ403-459 analysed by SDS-PAGE and Coomassie blue staining, following expression in SF21 cells, and DAC-tag-affinity purification and tag cleavage (see Materials and Methods). Bands migrating between 62- and 98-kDa protein markers are CUL3WT and CUL3Δ403-459, respectively, while the band below 17 kDa is Rbx1. The immunoblot for RBX1 is shown on the right. The band in CUL3WT denoted with * is co-purified insect cell CAND1, which was later removed by SEC. All protein identities were confirmed by mass spectrometry.


KLHL3 binding to CUL3WT and CUL3Δ403 - 459 is comparable as shown by pull - down assays with a KLHL3 resin generated from cross - linking anti - KLHL3 antibody to Protein G sepharose , and then binding saturating amounts of KLHL3 to the anti - KLHL3 resin ( denoted KLHL3R ) , the control resin ( ControlR ) is Protein G sepharose ( see Materials and Methods for further detail ) . CUL3WT or CUL3Δ403 - 459 was incubated with either KLHL3R or ControlR at 4 ° C for 1 h and then washed thrice with 1 × PBS , resin volumes were minimised , and SDS - Laemmli buffer was used to elute protein from resin prior to analysis by SDS - PAGE and staining with colloidal blue .

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

KLHL3 binding to CUL3WT and CUL3Δ403-459 is comparable as shown by pull-down assays with a KLHL3 resin generated from cross-linking anti-KLHL3 antibody to Protein G sepharose, and then binding saturating amounts of KLHL3 to the anti-KLHL3 resin (denoted KLHL3R), the control resin (ControlR) is Protein G sepharose (see Materials and Methods for further detail). CUL3WT or CUL3Δ403-459 was incubated with either KLHL3R or ControlR at 4°C for 1 h and then washed thrice with 1× PBS, resin volumes were minimised, and SDS-Laemmli buffer was used to elute protein from resin prior to analysis by SDS-PAGE and staining with colloidal blue.


UBE2D3 ˜ ubiquitin - release assays analysed by non - reducing SDS - PAGE and immunodetection . His - tagged UBE2D3 was charged with ubiquitin by incubation with ATP and UAE , and charging was stopped by ATP depletion with apyrase . No CUL3 ( negative control , lane 1 ) , CUL3WT or CUL3Δ403 - 459 was then added to the charged E2 and the activity of the RING domain , RBX1 , monitored by the hydrolysis of ubiquitin from UBE2D3 , visualised by immunodetection with anti - His antibody ( upper panel , " ˜ " denotes thioester bond ) . The lower panel is an immunoblot for CUL3 , showing equivalent amount of both CUL3WT and CUL3Δ403 - 459 was added to the reaction mix ; over time , the appearance of higher molecular weight bands represents the modification of CUL3 by covalent attachment of ubiquitin . The right two lanes are control lanes incubated for 10 min with E2 ˜ Ubi but then treated with reducing agent prior to SDS - PAGE .

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

UBE2D3˜ubiquitin-release assays analysed by non-reducing SDS-PAGE and immunodetection. His-tagged UBE2D3 was charged with ubiquitin by incubation with ATP and UAE, and charging was stopped by ATP depletion with apyrase. No CUL3 (negative control, lane 1), CUL3WT or CUL3Δ403-459 was then added to the charged E2 and the activity of the RING domain, RBX1, monitored by the hydrolysis of ubiquitin from UBE2D3, visualised by immunodetection with anti-His antibody (upper panel, "˜" denotes thioester bond). The lower panel is an immunoblot for CUL3, showing equivalent amount of both CUL3WT and CUL3Δ403-459 was added to the reaction mix; over time, the appearance of higher molecular weight bands represents the modification of CUL3 by covalent attachment of ubiquitin. The right two lanes are control lanes incubated for 10 min with E2˜Ubi but then treated with reducing agent prior to SDS-PAGE.


Structural model of CUL3WT based on the structure of CUL1 ( PDB : 1LDK ) , highlighting the residues deleted in the PHA2E mutant which are located close to the N - terminal domain ( NTD ) and C - terminal domain ( CTD ) border , using Phyre2 and Chimera ( see Materials and Methods ) . The NTD is coloured purple , the CTD cyan , and residues 403 - 459 orange .

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

Structural model of CUL3WT based on the structure of CUL1 (PDB:1LDK), highlighting the residues deleted in the PHA2E mutant which are located close to the N-terminal domain (NTD) and C-terminal domain (CTD) border, using Phyre2 and Chimera (see Materials and Methods). The NTD is coloured purple, the CTD cyan, and residues 403-459 orange.


Time course of in vitro neddylation reactions . Purified NAE , UBE2M and Nedd8 were mixed with either CUL3WT or CUL3Δ403 - 459 and incubated at 30 ° C for the time indicated . Due to the covalent attachment of Nedd8 ( 8 . 6 kDa ) , modified proteins have decreased mobility . The band shift observed for CUL3WT ( lane 1 versus lane 2 ) is representative of a single Nedd8 modification , while the multiple bands in the CUL3Δ403 - 459 reaction reflect the attachment of up to three Nedd8 molecules ( lane 5 versus lane 8 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 course of in vitro neddylation reactions. Purified NAE, UBE2M and Nedd8 were mixed with either CUL3WT or CUL3Δ403-459 and incubated at 30°C for the time indicated. Due to the covalent attachment of Nedd8 (8.6 kDa), modified proteins have decreased mobility. The band shift observed for CUL3WT (lane 1 versus lane 2) is representative of a single Nedd8 modification, while the multiple bands in the CUL3Δ403-459 reaction reflect the attachment of up to three Nedd8 molecules (lane 5 versus lane 8).


HEK - 293 cells over - expressing either FLAG - CUL3WT or FLAG - CUL3Δ403 - 459 were immunoprecipitated with M2 Flag - binding sepharose , in the presence of a deneddylase inhibitor ( OPT ) . Extract , cells were lysed in the presence of OPT and clarified by centrifugation . IP : M2 , clarified supernatant samples were incubated for 1 h at 4 ° C with M2 ( anti - Flag ) sepharose , then washed with PBS and eluted with SDS - Laemmli buffer , prior to SDS - PAGE and immunodetection with anti - FLAG antibody . Bands are labelled as follows : 1 . CUL3WT , 2 . CUL3WT - N8 , 3 . CUL3Δ403 - 459 , 4 . CUL3Δ403 - 459 - N8 , 5 . CUL3Δ403 - 459 - 2 ( N8 ) .

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

HEK-293 cells over-expressing either FLAG-CUL3WT or FLAG-CUL3Δ403-459 were immunoprecipitated with M2 Flag-binding sepharose, in the presence of a deneddylase inhibitor (OPT). Extract, cells were lysed in the presence of OPT and clarified by centrifugation. IP:M2, clarified supernatant samples were incubated for 1 h at 4°C with M2 (anti-Flag) sepharose, then washed with PBS and eluted with SDS-Laemmli buffer, prior to SDS-PAGE and immunodetection with anti-FLAG antibody. Bands are labelled as follows: 1. CUL3WT, 2. CUL3WT-N8, 3. CUL3Δ403-459, 4. CUL3Δ403-459-N8, 5. CUL3Δ403-459-2(N8).


Neddylation in HEK - 293 cells over - expressing FLAG - CUL3WT or FLAG - CUL3Δ403 - 459 was blocked by treatment with a Nedd8 - E1 - enzyme inhibitor , 3 μM MLN4924 ( Millennium Pharmaceuticals ) , in cells over - expressing FLAG - CUL3WT and FLAG - CUL3Δ403 - 459 respectively . Cell extracts were sampled over time in the presence of OPT to prevent de - neddylation and subjected to SDS - PAGE and immunodetection of the FLAG tag .

O O O O O O O O O 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

Neddylation in HEK-293 cells over-expressing FLAG-CUL3WT or FLAG-CUL3Δ403-459 was blocked by treatment with a Nedd8-E1-enzyme inhibitor, 3 μM MLN4924 (Millennium Pharmaceuticals), in cells over-expressing FLAG-CUL3WT and FLAG-CUL3Δ403-459 respectively. Cell extracts were sampled over time in the presence of OPT to prevent de-neddylation and subjected to SDS-PAGE and immunodetection of the FLAG tag.


FLAG - CUL3WT or FLAG - CUL3Δ403 - 459 was immunoprecipitated with M2 - sepharose as described in ( F ) . + MLN4924 indicates cell culture media supplementation with 3 μM MLN4924 for 5 h prior to cell lysis to achieve complete deneddylation of CUL3 . Following SDS - PAGE , immunoblotting with COP9 Signalosome ( CSN ) or CAND1 antibodies determined the interaction between these Cullin regulators and FLAG - CUL3WT and FLAG - CUL3Δ403 - 459 , respectively .

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 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

FLAG-CUL3WT or FLAG-CUL3Δ403-459 was immunoprecipitated with M2-sepharose as described in (F). +MLN4924 indicates cell culture media supplementation with 3 μM MLN4924 for 5 h prior to cell lysis to achieve complete deneddylation of CUL3. Following SDS-PAGE, immunoblotting with COP9 Signalosome (CSN) or CAND1 antibodies determined the interaction between these Cullin regulators and FLAG-CUL3WT and FLAG-CUL3Δ403-459, respectively.


GST - CAND1 pull - down assays with CUL3 proteins . Purified GST or GST - CAND1 immobilised on glutathione - sepharose was mixed with either purified CUL3WT or CUL3Δ403 - 459 and incubated at 4 ° C for 1 h . The sepharose was then washed thrice and eluted from the resin by incubation with SDS - Laemmli buffer prior to SDS - PAGE and Coomassie staining . CUL3WT but not CUL3Δ403 - 459 interacts with CAND1 .

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

GST-CAND1 pull-down assays with CUL3 proteins. Purified GST or GST-CAND1 immobilised on glutathione-sepharose was mixed with either purified CUL3WT or CUL3Δ403-459 and incubated at 4°C for 1 h. The sepharose was then washed thrice and eluted from the resin by incubation with SDS-Laemmli buffer prior to SDS-PAGE and Coomassie staining. CUL3WT but not CUL3Δ403-459 interacts with CAND1.


A - D In vitro ubiquitylation assays with purified proteins . As ubiquitin is covalently attached to the substrate lysine residue , the appearance of higher molecular weight protein bands reflects the modification of the protein with ubiquitin . All assays contain purified UBE1 , UBE2D3 , ubiquitin , 0 . 1 mM ATP , 1 mM MgCl2 and were buffered in 50 mM HEPES , 150 mM NaCl and incubated at 30 ° C for the time indicated . Reactions were stopped by the addition of SDS - Laemmli buffer to a concentration of 1 × . SDS - PAGE , staining with Coomassie blue , or detection with the indicated antibody following immunoblotting enabled the visualisation of ubiquitylation . ( A ) to determine the relative modification of KLHL3 by CUL3WT and CUL3Δ403 - 459 , KLHL3 was included into the reaction at 2 × molar concentration over CUL3WT and CUL3Δ403 - 459 . ( B , C ) Reactions serve to determine basal auto - ubiquitylation of CUL3WT or CUL3Δ403 - 459 and do not contain KLHL3 or other potential substrate proteins . Lysine residues on CUL3WT or CUL3Δ403 - 459 act as the substrate . ( B ) High molecular weight bands reflect ubiquitin chain linkages on CUL3 or the multiple mono - ubiquitylation of a number of CUL3 lysine residues . ( C ) Activity assays contain methylated ubiquitin , a form of ubiquitin incapable of forming ubiquitin chains ; as such , higher band shifts reflect the attachment of mono - ubiquitin to one more lysine residue on CUL3WT or CUL3Δ403 - 459 , respectively . ( D ) Activity assay performed as in ( C ) with methyl - ubiquitin , the boxes shown on the gel are indicative of the gel pieces excised for mass spectrometry analysis in ( F ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-D In vitro ubiquitylation assays with purified proteins. As ubiquitin is covalently attached to the substrate lysine residue, the appearance of higher molecular weight protein bands reflects the modification of the protein with ubiquitin. All assays contain purified UBE1, UBE2D3, ubiquitin, 0.1 mM ATP, 1 mM MgCl2 and were buffered in 50 mM HEPES, 150 mM NaCl and incubated at 30°C for the time indicated. Reactions were stopped by the addition of SDS-Laemmli buffer to a concentration of 1×. SDS-PAGE, staining with Coomassie blue, or detection with the indicated antibody following immunoblotting enabled the visualisation of ubiquitylation. (A) to determine the relative modification of KLHL3 by CUL3WT and CUL3Δ403-459, KLHL3 was included into the reaction at 2× molar concentration over CUL3WT and CUL3Δ403-459. (B, C) Reactions serve to determine basal auto-ubiquitylation of CUL3WT or CUL3Δ403-459 and do not contain KLHL3 or other potential substrate proteins. Lysine residues on CUL3WT or CUL3Δ403-459 act as the substrate. (B) High molecular weight bands reflect ubiquitin chain linkages on CUL3 or the multiple mono-ubiquitylation of a number of CUL3 lysine residues. (C) Activity assays contain methylated ubiquitin, a form of ubiquitin incapable of forming ubiquitin chains; as such, higher band shifts reflect the attachment of mono-ubiquitin to one more lysine residue on CUL3WT or CUL3Δ403-459, respectively. (D) Activity assay performed as in (C) with methyl-ubiquitin, the boxes shown on the gel are indicative of the gel pieces excised for mass spectrometry analysis in (F).


F Structural docking model of CUL3Δ403 - 459 based on the structure of full - length CUL1 ( 1LDK ) using Chimera ( see Materials and Methods ) . The NTD is coloured green , while the CTD is red , and the four panels sample possible positioning of the CTD relative to the NTD in CUL3Δ403 - 459 , assuming full flexibility of the linker after deletion of three helices encoded by exon 9 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Structural docking model of CUL3Δ403-459 based on the structure of full-length CUL1 (1LDK) using Chimera (see Materials and Methods). The NTD is coloured green, while the CTD is red, and the four panels sample possible positioning of the CTD relative to the NTD in CUL3Δ403-459, assuming full flexibility of the linker after deletion of three helices encoded by exon 9.


A - C In vitro ubiquitylation assays were performed as described in Figure 2 , but with the addition of immunoprecipitatedWNK1 in ( A ) . In vitro ubiquitylation assays with purified proteins . As ubiquitin is covalently attached to the substrate lysine residue , the appearance of higher molecular weight protein bands reflects the modification of the protein with ubiquitin . All assays contain purified UBE1 , UBE2D3 , ubiquitin , 0 . 1 mM ATP , 1 mM MgCl2 and were buffered in 50 mM HEPES , 150 mM NaCl and incubated at 30 ° C for the time indicated . Reactions were stopped by the addition of SDS - Laemmli buffer to a concentration of 1 × . SDS - PAGE , staining with Coomassie blue , or detection with the indicated antibody following immunoblotting enabled the visualisation of ubiquitylation .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 In vitro ubiquitylation assays were performed as described in Figure 2, but with the addition of immunoprecipitatedWNK1 in (A). In vitro ubiquitylation assays with purified proteins. As ubiquitin is covalently attached to the substrate lysine residue, the appearance of higher molecular weight protein bands reflects the modification of the protein with ubiquitin. All assays contain purified UBE1, UBE2D3, ubiquitin, 0.1 mM ATP, 1 mM MgCl2 and were buffered in 50 mM HEPES, 150 mM NaCl and incubated at 30°C for the time indicated. Reactions were stopped by the addition of SDS-Laemmli buffer to a concentration of 1×. SDS-PAGE, staining with Coomassie blue, or detection with the indicated antibody following immunoblotting enabled the visualisation of ubiquitylation.


A - C In vitro ubiquitylation assays were performed as described in Figure 2 , but with the addition of immunoprecipitated over - expressed FLAG - WNK4 in ( B , C ) ( see Materials and Methods ) . The WNK kinases are modified by CUL3WT - KLHL3 , with the higher molecular weight smear observed in anti - WNK1 and anti - FLAG panels representative of multiple ubiquitin molecules being covalently attached to the WNK protein . CUL3Δ403 - 459 is unable to modify WNKs . Samples from the same assay reactions were divided to allow immunodetection of the different protein components modified within the same assay reaction . ( C ) CUL3WT , CUL3Δ403 - 459 and an equimolar solution CUL3WT : CUL3Δ403 - 459 ( 1 : 1 Mix ) were incubated with KLHL3 and immunoprecipitatedFLAG - WNK4 in ubiquitylation reactions to determine the influence of CUL3Δ403 - 459 on the ubiquitylation activity of CUL3WT . Notably , the presence of CUL3Δ403 - 459 does not inhibit WNK ubiquitylation by CUL3WT . In vitro ubiquitylation assays with purified proteins . As ubiquitin is covalently attached to the substrate lysine residue , the appearance of higher molecular weight protein bands reflects the modification of the protein with ubiquitin . All assays contain purified UBE1 , UBE2D3 , ubiquitin , 0 . 1 mM ATP , 1 mM MgCl2 and were buffered in 50 mM HEPES , 150 mM NaCl and incubated at 30 ° C for the time indicated . Reactions were stopped by the addition of SDS - Laemmli buffer to a concentration of 1 × . SDS - PAGE , staining with Coomassie blue , or detection with the indicated antibody following immunoblotting enabled the visualisation of ubiquitylation .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 In vitro ubiquitylation assays were performed as described in Figure 2, but with the addition of immunoprecipitated over-expressed FLAG-WNK4 in (B, C) (see Materials and Methods). The WNK kinases are modified by CUL3WT-KLHL3, with the higher molecular weight smear observed in anti-WNK1 and anti-FLAG panels representative of multiple ubiquitin molecules being covalently attached to the WNK protein. CUL3Δ403-459 is unable to modify WNKs. Samples from the same assay reactions were divided to allow immunodetection of the different protein components modified within the same assay reaction. (C) CUL3WT, CUL3Δ403-459 and an equimolar solution CUL3WT:CUL3Δ403-459 (1:1 Mix) were incubated with KLHL3 and immunoprecipitatedFLAG-WNK4 in ubiquitylation reactions to determine the influence of CUL3Δ403-459 on the ubiquitylation activity of CUL3WT. Notably, the presence of CUL3Δ403-459 does not inhibit WNK ubiquitylation by CUL3WT.In vitro ubiquitylation assays with purified proteins. As ubiquitin is covalently attached to the substrate lysine residue, the appearance of higher molecular weight protein bands reflects the modification of the protein with ubiquitin. All assays contain purified UBE1, UBE2D3, ubiquitin, 0.1 mM ATP, 1 mM MgCl2 and were buffered in 50 mM HEPES, 150 mM NaCl and incubated at 30°C for the time indicated. Reactions were stopped by the addition of SDS-Laemmli buffer to a concentration of 1×. SDS-PAGE, staining with Coomassie blue, or detection with the indicated antibody following immunoblotting enabled the visualisation of ubiquitylation.


Western blot of whole kidney lysates from mice culled after a minimum 4 - h fast . Following exsanguination after surgery , mouse tissues were rapidly harvested and stored , samples were then homogenised , clarified and quantified prior to SDS - PAGE . Immunodetection with the antibodies shown highlights elevated signalling through the WNK kinase pathway in CUL3WT / Δ403 - 459 versus CUL3WT mice . The lowest panel is an anti - CUL3 immunoprecipitation of the kidney lysate samples , whereby the CUL3 antibody was cross - linked to Protein G sepharose and used to affinity purify CUL3WT and CUL3Δ403 - 459 from kidney lysates . The samples were incubated together overnight to allow deneddylation of CUL3 proteins to occur and maximise CUL3 binding to the anti - CUL3 resin . Samples were thoroughly washed and eluted from the resin prior to SDS - PAGE and immunodetection for CUL3 . The IP highlights that CUL3Δ403 - 459 is indeed present within the kidney lysate .

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

Western blot of whole kidney lysates from mice culled after a minimum 4-h fast. Following exsanguination after surgery, mouse tissues were rapidly harvested and stored, samples were then homogenised, clarified and quantified prior to SDS-PAGE. Immunodetection with the antibodies shown highlights elevated signalling through the WNK kinase pathway in CUL3WT/Δ403-459 versus CUL3WT mice. The lowest panel is an anti-CUL3 immunoprecipitation of the kidney lysate samples, whereby the CUL3 antibody was cross-linked to Protein G sepharose and used to affinity purify CUL3WT and CUL3Δ403-459 from kidney lysates. The samples were incubated together overnight to allow deneddylation of CUL3 proteins to occur and maximise CUL3 binding to the anti-CUL3 resin. Samples were thoroughly washed and eluted from the resin prior to SDS-PAGE and immunodetection for CUL3. The IP highlights that CUL3Δ403-459 is indeed present within the kidney lysate.


Fresh frozen kidney tissues from transplant patient and donor cadavers were homogenised , clarified and quantified prior to SDS - PAGE . Western blot analysis confirmed the expression of KLHL3 and CUL3 in normal healthy human kidneys .

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

Fresh frozen kidney tissues from transplant patient and donor cadavers were homogenised, clarified and quantified prior to SDS-PAGE. Western blot analysis confirmed the expression of KLHL3 and CUL3 in normal healthy human kidneys.


Plasma aldosterone after a minimum 4 - h fast was calculated by HTRF ( homogeneous time - resolved fluorescence ) aldosterone assay . The average aldosterone level per mouse was calculated from duplicate samples run in parallel on the assay . Blood was rapidly harvested in heparin - coated plasma extraction tubes following exsanguination after surgery , and samples were snap - frozen for storage . A 58 % increase in aldosterone was detected in CUL3WT / Δ403 - 459 versus CUL3WT mice ( * P = 0 . 0245 ) . Two - tailed unpaired Student ' s t - test ; data are mean ± SEM .

O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_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 B-MEASURED_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

Plasma aldosterone after a minimum 4-h fast was calculated by HTRF (homogeneous time-resolved fluorescence) aldosterone assay. The average aldosterone level per mouse was calculated from duplicate samples run in parallel on the assay. Blood was rapidly harvested in heparin-coated plasma extraction tubes following exsanguination after surgery, and samples were snap-frozen for storage. A 58% increase in aldosterone was detected in CUL3WT/Δ403-459 versus CUL3WT mice (*P = 0.0245). Two-tailed unpaired Student's t-test; data are mean ± SEM.


Arterial blood biochemistries after a minimum 4 - h fast . Under anaesthesia , the right carotid artery was cannulated to minimise atmospheric exposure of samples collected for iSTATbloodgas and electrolyte measurements . CUL3WT / Δ403 - 459mice present with abnormal electrolyte homoeostasis compared to CUL3WTmice , exhibiting hyperkalaemia ( * * * P = 0 . 0004 ) and hyperchloraemia ( * * * P = 9 . 5 × 10 − 5 ) with a compensated metabolic acidosis ( P = 0 . 7766 ) , marked by a decrease in bicarbonate ( ) ( * * * P = 3 . 4 × 10 − 5 ) , base excess ( BE ) ( * * * P = 9 . 1 × 10 − 5 ) and partial pressure of carbon dioxide ( pCO2 ) ( * * * P = 0 . 0038 ) . Two - tailed unpaired Student ' s t - test ; data 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 B-MEASURED_VAR O O O O O O O O O O B-MEASURED_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 B-MEASURED_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 B-MEASURED_VAR I-MEASURED_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

Arterial blood biochemistries after a minimum 4-h fast. Under anaesthesia, the right carotid artery was cannulated to minimise atmospheric exposure of samples collected for iSTATbloodgas and electrolyte measurements. CUL3WT/Δ403-459mice present with abnormal electrolyte homoeostasis compared to CUL3WTmice, exhibiting hyperkalaemia (***P = 0.0004) and hyperchloraemia (***P = 9.5 × 10−5) with a compensated metabolic acidosis (P = 0.7766), marked by a decrease in bicarbonate () (***P = 3.4 × 10−5), base excess (BE) (***P = 9.1 × 10−5) and partial pressure of carbon dioxide (pCO2) (***P = 0.0038). Two-tailed unpaired Student's t-test; data are mean ± SEM.


Representative pseudocoloured maximum - intensity z projections of immunofluorescence stained kidney sections showing the distribution of the WNK / SPAK pathway components between CUL3WT / Δ403 - 459 and CUL3WTmice at a minimum 4 - h fasting baseline ( n = 4 per genotype ) . CUL3 and KLHL3 are comparable between genotypes , with significantly higher levels of CUL3 in the proximal convoluted tubule ( PCT ) compared to the weak staining in distal convoluted tubule ( DCT ) and thick ascending limb of the loop of Henle ( TAL ) , while KLHL3 shows higher expression in the DCT / TAL cytosol with staining of the PCT confined to the apical membrane . Total ( t ) and phospho ( p ) NCC Thr44 and SPAK Thr243 show increased apical membrane expression in the parvalbumin ( PVALB ) - positive early and late ( PVALB - negative ) DCT of CUL3WT / Δ403 - 459mice . Unexpectedly , the increased levels of WNK4 and SPAK resulted in the formation of discrete puncta specifically in the DCT of CUL3WT / Δ403 - 459mice . It is possible that the autophagy - lysosomal system may attempt to compensate and degrade these excess proteins although the WNK4 puncta do not colocalise with the lysosomal marker LAMP1 . 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 pseudocoloured maximum-intensity z projections of immunofluorescence stained kidney sections showing the distribution of the WNK/SPAK pathway components between CUL3WT/Δ403-459 and CUL3WTmice at a minimum 4-h fasting baseline (n = 4 per genotype). CUL3 and KLHL3 are comparable between genotypes, with significantly higher levels of CUL3 in the proximal convoluted tubule (PCT) compared to the weak staining in distal convoluted tubule (DCT) and thick ascending limb of the loop of Henle (TAL), while KLHL3 shows higher expression in the DCT/TAL cytosol with staining of the PCT confined to the apical membrane. Total (t) and phospho (p) NCC Thr44 and SPAK Thr243 show increased apical membrane expression in the parvalbumin (PVALB)-positive early and late (PVALB-negative) DCT of CUL3WT/Δ403-459mice. Unexpectedly, the increased levels of WNK4 and SPAK resulted in the formation of discrete puncta specifically in the DCT of CUL3WT/Δ403-459mice. It is possible that the autophagy-lysosomal system may attempt to compensate and degrade these excess proteins although the WNK4 puncta do not colocalise with the lysosomal marker LAMP1. Scale bar, 20 μm.


Representative immunohistochemistry staining of KLHL3 and CUL3 in humankidney sections ( n = 6 ) . KLHL3 shows preferential DCT / TAL cytosolic staining similar to the mouse despite no evidence of PCT apical staining as seen in ( A ) . CUL3 exhibits preferential basolateral cytosolic staining of the PCT with similarly low levels of diffuse staining in the DCT / TAL to that of the mouse . Scale bar , 100 μm .

Representative immunohistochemistry staining of KLHL3 and CUL3 in humankidney sections (n = 6). KLHL3 shows preferential DCT/TAL cytosolic staining similar to the mouse despite no evidence of PCT apical staining as seen in (A). CUL3 exhibits preferential basolateral cytosolic staining of the PCT with similarly low levels of diffuse staining in the DCT/TAL to that of the mouse. Scale bar, 100 μm.


Continuous blood pressure measurements were taken in mice under general anaesthesia with thermostatically controlled body temperature , right carotid artery pressure transducer catheterisation and ECG Lead II heart rate monitoring . CUL3WT / Δ403 - 459 mice have elevated systolic ( SBP ) ( * * * P = 1 × 10 − 12 ) and diastolic ( DBP ) blood pressure ( * * * P = 8 . 5 × 10 − 8 ) , although they also present with 7 % lower heart rates ( * P = 0 . 0485 ) when compared to CUL3WT mice . Two - tailed unpaired Student ' s t - test ; data 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Continuous blood pressure measurements were taken in mice under general anaesthesia with thermostatically controlled body temperature, right carotid artery pressure transducer catheterisation and ECG Lead II heart rate monitoring. CUL3WT/Δ403-459 mice have elevated systolic (SBP) (***P = 1 × 10−12) and diastolic (DBP) blood pressure (***P = 8.5 × 10−8), although they also present with 7% lower heart rates (*P = 0.0485) when compared to CUL3WT mice. Two-tailed unpaired Student's t-test; data are mean ± SEM.


Pulse waveform analysis of blood pressure traces obtained in ( A ) reveals that CUL3WT / Δ403 - 459 mice have an increased pulse pressure ( PP ) [ SBP - DBP ] ( * * * P = 2 . 1 × 10 − 10 ) , augmentation pressure ( AP ) [ SBP - anacrotic notch ( AN ) pressure ] ( * * * P = 3 . 5 × 10 − 6 ) , dicrotic notch ( DN ) pressure ( * * * P = 4 . 7 × 10 − 13 ) and mean arterial pressure ( MAP ) [ 1 / 3 × SBP + 2 / 3 × DBP ] ( * * * P = 1 . 3 × 10 − 9 ) . This hypertensive phenotype is in part due to changes in vascular contractility in CUL3WT / Δ403 - 459 mice as evidenced by their higher augmentation index ( AIx ) [ AP / PP ] ( * * P = 0 . 0096 ) , a marker of arterial stiffness , and is further supported by an increase in their diastolic pressure decay time constant ( τbourgeois ) [ 1 / slope of diastolic pressure decay ; measured 30 ms after DN and 20 ms before end DBP ] ( * * P = 0 . 0083 ) , a surrogate marker of increased vascular resistance . Two - tailed unpaired Student ' s t - test ; data are mean ±

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

Pulse waveform analysis of blood pressure traces obtained in (A) reveals that CUL3WT/Δ403-459 mice have an increased pulse pressure (PP) [SBP - DBP] (***P = 2.1 × 10−10), augmentation pressure (AP) [SBP - anacrotic notch (AN) pressure] (***P = 3.5 × 10−6), dicrotic notch (DN) pressure (***P = 4.7 × 10−13) and mean arterial pressure (MAP) [1/3 × SBP + 2/3 × DBP] (***P = 1.3 × 10−9). This hypertensive phenotype is in part due to changes in vascular contractility in CUL3WT/Δ403-459 mice as evidenced by their higher augmentation index (AIx) [AP/PP] (**P = 0.0096), a marker of arterial stiffness, and is further supported by an increase in their diastolic pressure decay time constant (τbourgeois) [1/slope of diastolic pressure decay; measured 30 ms after DN and 20 ms before end DBP] (**P = 0.0083), a surrogate marker of increased vascular resistance. Two-tailed unpaired Student's t-test; data are mean ± SEM


In vivo dose - responses to phenylephrine and angiotensin II . After baseline measurements were obtained from mice in ( A ) , the right external jugular vein was cannulated for administration of bolus doses of vasopressors in increasing half - log steps . Dose - response curves were generated and data analysed using a logistical function to determine the estimated dose producing half - maximal response ( ED50 ) and maximum response ( Emax ) . The fitted Emax for phenylephrine was increased in CUL3WT / Δ403 - 459 versus CUL3WT ( 183 . 9 ± 2 . 5 versus 164 . 9 ± 1 . 4 mmHg ) ( * * * P = 1 × 10 − 6 ) indicating an increased vasoconstrictor response to adrenergic stimulation elevating systolic blood pressure substantially above the CUL3WT maximum . Similarly , the fitted Emax for angiotensin II stimulation was higher in CUL3WT / Δ403 - 459 versus CUL3WT ( 155 . 5 ± 1 . 8 versus 138 . 3 ± 2 . 2 mmHg ) ( * * * P = 1 . 7 × 10 − 6 ) . However , there was no change between CUL3WT / Δ403 - 459 versus CUL3WT sensitivity ( as measured by ED50 ) to phenylephrine ( 36 . 6 ± 5 . 1 versus 31 . 4 ± 2 . 8 μg / kg bw ) ( P = 0 . 3778 ) or angiotensin II ( 0 . 77 ± 0 . 16 versus 0 . 93 ± 0 . 13 μg / kg bw ) ( P = 0 . 4401 ) . Two - tailed unpaired Student ' s t - test ; data are mean ± SEM .

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 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 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

In vivo dose-responses to phenylephrine and angiotensin II. After baseline measurements were obtained from mice in (A), the right external jugular vein was cannulated for administration of bolus doses of vasopressors in increasing half-log steps. Dose-response curves were generated and data analysed using a logistical function to determine the estimated dose producing half-maximal response (ED50) and maximum response (Emax). The fitted Emax for phenylephrine was increased in CUL3WT/Δ403-459 versus CUL3WT (183.9 ± 2.5 versus 164.9 ± 1.4 mmHg) (***P = 1 × 10−6) indicating an increased vasoconstrictor response to adrenergic stimulation elevating systolic blood pressure substantially above the CUL3WT maximum. Similarly, the fitted Emax for angiotensin II stimulation was higher in CUL3WT/Δ403-459 versus CUL3WT (155.5 ± 1.8 versus 138.3 ± 2.2 mmHg) (***P = 1.7 × 10−6). However, there was no change between CUL3WT/Δ403-459 versus CUL3WT sensitivity (as measured by ED50) to phenylephrine (36.6 ± 5.1 versus 31.4 ± 2.8 μg/kg bw) (P = 0.3778) or angiotensin II (0.77 ± 0.16 versus 0.93 ± 0.13 μg/kg bw) (P = 0.4401). Two-tailed unpaired Student's t-test; data are mean ± SEM.


Western blot of tunica media - intimathoracic aorta lysates from mice culled after a minimum 4 - h fast . Following exsanguination after surgery , mouse tissues were rapidly harvested and the tunica adventitia removed before storage , and later , the samples were homogenised , clarified and quantified prior to SDS - PAGE . Western blot analysis confirmed the expression of KLHL3 and CUL3 . Similar to the kidney , the aorta of CUL3WT / Δ403 - 459 showed slightly lower levels of CUL3 compared to CUL3WT without any change in KLHL3 levels .

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

Western blot of tunica media-intimathoracic aorta lysates from mice culled after a minimum 4-h fast. Following exsanguination after surgery, mouse tissues were rapidly harvested and the tunica adventitia removed before storage, and later, the samples were homogenised, clarified and quantified prior to SDS-PAGE. Western blot analysis confirmed the expression of KLHL3 and CUL3. Similar to the kidney, the aorta of CUL3WT/Δ403-459 showed slightly lower levels of CUL3 compared to CUL3WT without any change in KLHL3 levels.


Fresh frozen thoracic aorta tissues from donor cadavers were homogenised , clarified and quantified prior to SDS - PAGE . Western blot analysis confirmed the expression of KLHL3 and CUL3 in normal healthy human aorta .

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

Fresh frozen thoracic aorta tissues from donor cadavers were homogenised, clarified and quantified prior to SDS-PAGE. Western blot analysis confirmed the expression of KLHL3 and CUL3 in normal healthy human aorta.


Representative maximum - intensity z projections of immunofluorescence stained thoracic aorta sections showing the distribution of CUL3 and KLHL3 between CUL3WT / Δ403 - 459 and CUL3WT mice at a minimum 4 - h fasting baseline ( n = 4 per genotype ) . CUL3 and KLHL3 localisation is comparable between genotypes . The highest levels were detected in the vascular smooth muscle cells and endothelium , with a minimal expression in the perivascular adipose tissue of the adventitia . Scale bar , 50 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

Representative maximum-intensity z projections of immunofluorescence stained thoracic aorta sections showing the distribution of CUL3 and KLHL3 between CUL3WT/Δ403-459 and CUL3WT mice at a minimum 4-h fasting baseline (n = 4 per genotype). CUL3 and KLHL3 localisation is comparable between genotypes. The highest levels were detected in the vascular smooth muscle cells and endothelium, with a minimal expression in the perivascular adipose tissue of the adventitia. Scale bar, 50 μm.


Representative immunohistochemistry staining of KLHL3 and CUL3 in humanthoracic aorta sections ( n = 6 ) . Similar to the mouse staining seen in ( C ) , vascular smooth muscle cells in the tunica media of the aortic wall are positive for KLHL3 and CUL3 . Scale bar , 50 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

Representative immunohistochemistry staining of KLHL3 and CUL3 in humanthoracic aorta sections (n = 6). Similar to the mouse staining seen in (C), vascular smooth muscle cells in the tunica media of the aortic wall are positive for KLHL3 and CUL3. Scale bar, 50 μm.


Morphometric analysis of thoracic aortae reveals vascular remodelling in CUL3WT / Δ403 - 459 mice . There is an increase of ˜ 21 % in the vessel wall intima - media thickness ( demarcated by arrows ) of CUL3WT / Δ403 - 459 compared to CUL3WT mice ( * * * P = 0 . 0003 ) . However , there is no change in the number of elastin laminae ( P = 0 . 1458 ) and therefore no increase in the number of medial muscle layers between genotypes . Two - tailed unpaired Student ' s t - test ; data 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Morphometric analysis of thoracic aortae reveals vascular remodelling in CUL3WT/Δ403-459 mice. There is an increase of ˜21% in the vessel wall intima-media thickness (demarcated by arrows) of CUL3WT/Δ403-459 compared to CUL3WT mice (***P = 0.0003). However, there is no change in the number of elastin laminae (P = 0.1458) and therefore no increase in the number of medial muscle layers between genotypes. Two-tailed unpaired Student's t-test; data are mean ± SEM.


A Normal lung cells ( IMR90 and MRC5 ) and lung cancer cells ( A549 and H1975 ) were treated with various concentrations of PM2 . 5 for 24 h , and cell viability was assessed by a trypan blue assay . The values are the mean ± SD of three 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 O O O O O O O O O O O O O O O O O

A Normal lung cells (IMR90 and MRC5) and lung cancer cells (A549 and H1975) were treated with various concentrations of PM2.5 for 24 h, and cell viability was assessed by a trypan blue assay. The values are the mean ± SD of three independent experiments.


B H1975 and A549 cells were treated with various concentrations of PM2 . 5 for 24 h , and their cell lysates were analyzed for phosphor - ERK ( pERK ) , phosphor - AKT ( pAKT ) , phosphor - STAT3 ( pSTAT3 ) , phosphor - EGFR ( pEGFR ) , ERK , AKT , STAT3 , and EGFR by Western blotting . β - actin served as the loading control . The results shown are from one of three similar experiments ( left panel ) . The relative expression levels of pEGFR , pAKT , pSTAT3 , and pERK were quantified by normalized with β - actin and are shown at the right panel . The values are the mean ± SD of three independent experiments . * p < 0 . 05 , as analyzed with one - sample t - test and compared with untreated cells .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 H1975 and A549 cells were treated with various concentrations of PM2.5 for 24 h, and their cell lysates were analyzed for phosphor-ERK (pERK), phosphor-AKT (pAKT), phosphor-STAT3 (pSTAT3), phosphor-EGFR (pEGFR), ERK, AKT, STAT3, and EGFR by Western blotting. β-actin served as the loading control. The results shown are from one of three similar experiments (left panel). The relative expression levels of pEGFR, pAKT, pSTAT3, and pERK were quantified by normalized with β-actin and are shown at the right panel. The values are the mean ± SD of three independent experiments. *p < 0.05, as analyzed with one-sample t-test and compared with untreated cells.


A , B H1975 and A549 cells were treated with PM2 . 5 at 50 μg / ml for 90 days , and the proliferation of the treated cells was assessed by trypan blue assays .

A, B H1975 and A549 cells were treated with PM2.5 at 50 μg/ml for 90 days, and the proliferation of the treated cells was assessed by trypan blue assays.