Representative images of anti - nuclear antibody ( ANA ) staining obtained with serum from 12 - month - old Prdm1ihCd2 / + or wild - type mice ( right ) , as detected by indirect immunofluorescence assay using HEp - 2 cells and an Alexa488 - conjugated anti - mouse IgG detection antibody . The serum of Faslgld / gld mice was used as positive control . The presence of ANA - IgG antibodies was analyzed for 22 Prdm1ihCd2 / + and 12 wild - type mice , as summarized in the pie - chart ( 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 O O O O O O O O O O O O O O O O O O O O O O O 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 images of anti-nuclear antibody (ANA) staining obtained with serum from 12-month-old Prdm1ihCd2/+ or wild-type mice (right), as detected by indirect immunofluorescence assay using HEp-2 cells and an Alexa488-conjugated anti-mouse IgG detection antibody. The serum of Faslgld/gld mice was used as positive control. The presence of ANA-IgG antibodies was analyzed for 22 Prdm1ihCd2/+ and 12 wild-type mice, as summarized in the pie-chart (right)


Periodic acid - Schiff ( PAS ) staining of paraffin - embedded kidney sections of Prdm1ihCd2 / + and wild - type mice at 12 months of age . The mean score of kidney pathology was determined for each mouse by evaluating 40 individual glomeruli to assess the severity of glomerulonephritis as shown in Table EV2 and described in the Appendix Supplementary Methods . Representative pictures of glomeruli of Prdm1ihCd2 / + kidneys with different pathology scores are shown to the left , and dot plots with average scores for male and female mice ( Table EV2 ) are shown to the right . Arrows denote intracapillary hyaline " thrombi " , asterisks indicate mesangial sclerosis with obscured capillary loops , and the arrowhead points to an obsolescent glomerulus with collapse of the glomerular tuft architecture . Statistical data ( A , C ) are shown as mean value with SEM and were analyzed by the Mann - Whitney test ( A ) or the Student " s t - test ( C ) ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Each dot

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

Periodic acid-Schiff (PAS) staining of paraffin-embedded kidney sections of Prdm1ihCd2/+ and wild-type mice at 12 months of age. The mean score of kidney pathology was determined for each mouse by evaluating 40 individual glomeruli to assess the severity of glomerulonephritis as shown in Table EV2 and described in the Appendix Supplementary Methods. Representative pictures of glomeruli of Prdm1ihCd2/+ kidneys with different pathology scores are shown to the left, and dot plots with average scores for male and female mice (Table EV2) are shown to the right. Arrows denote intracapillary hyaline "thrombi", asterisks indicate mesangial sclerosis with obscured capillary loops, and the arrowhead points to an obsolescent glomerulus with collapse of the glomerular tuft architecture. Statistical data (A,C) are shown as mean value with SEM and were analyzed by the Mann-Whitney test (A) or the Student"s t-test (C); *P < 0.05, **P < 0.01, ***P < 0.001. Each dot correspon


( A ) The intracellular distribution of DOR is modified by starvation . HeLa cells were transiently transfected with DOR and incubated for 1 h with DMEM ( basal ) , HBSS ( starvation ) , 50 μM chloroquine or 10 mM 3MA . The intracellular localization of DOR was analysed by immunofluorescence and is shown in red . The nuclei are shown in blue ( DAPI staining ) . Scale bars , 10 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 B-CONTROLLED_VAR 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

(A) The intracellular distribution of DOR is modified by starvation. HeLa cells were transiently transfected with DOR and incubated for 1 h with DMEM (basal), HBSS (starvation), 50 μM chloroquine or 10 mM 3MA. The intracellular localization of DOR was analysed by immunofluorescence and is shown in red. The nuclei are shown in blue (DAPI staining). Scale bars, 10 μm.


( B ) Endogenous DOR leaves the nucleus under starvation . C2C12 myoblasts were incubated for 30 min at 37 ° C with DMEM ( basal ) or HBSS ( starvation ) . DOR is shown in red and nuclei are in blue ( DAPI staining ) . Scale bars , 5 μm .

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

(B) Endogenous DOR leaves the nucleus under starvation. C2C12 myoblasts were incubated for 30 min at 37°C with DMEM (basal) or HBSS (starvation). DOR is shown in red and nuclei are in blue (DAPI staining). Scale bars, 5 μm.


( C , D ) De ‐ activation of autophagy returns DOR to the nucleus . HeLa cells were transiently transfected with DOR and incubated for 1 h with DMEM ( basal ) or HBSS ( starvation ) . Cells were then incubated with normal DMEM for an additional hour ( starvation + GM ) . The intracellular distribution of DOR was determined by immunofluorescence . DOR is shown in red and the nuclei are in blue . Scale bars , 5 μm . Cell lysates were also obtained and western blot assays were performed with specific antibodies . 3MA , 3 ‐ methyladenine ; DAPI , 4 ′ , 6 ‐ diamidino ‐ 2 ‐ phenylindole ; DMEM , Dulbecco ' s modified Eagle medium ; DOR , diabetes ‐ and obesity ‐ regulated gene ; HBSS , Hank ' s balanced salt solution ; LC3 , microtubule ‐ associated protein 1A / 1B ‐ light chain 3 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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,D) De‐activation of autophagy returns DOR to the nucleus. HeLa cells were transiently transfected with DOR and incubated for 1 h with DMEM (basal) or HBSS (starvation). Cells were then incubated with normal DMEM for an additional hour (starvation+GM). The intracellular distribution of DOR was determined by immunofluorescence. DOR is shown in red and the nuclei are in blue. Scale bars, 5 μm. Cell lysates were also obtained and western blot assays were performed with specific antibodies. 3MA, 3‐methyladenine; DAPI, 4′,6‐diamidino‐2‐phenylindole; DMEM, Dulbecco's modified Eagle medium; DOR, diabetes‐ and obesity‐regulated gene; HBSS, Hank's balanced salt solution; LC3, microtubule‐associated protein 1A/1B‐light chain 3.


DOR localizes to autophagosomes when autophagy is activated . ( A ) Confocal images of HeLa cells transiently transfected with DOR and GFP ‐ LC3 , and incubated for 1h with DMEM , HBSS ( starvation ) , 2 μM rapamycin or HBSS containing 50 μM chloroquine . Nuclei were labelled with DAPI . Scale bars , 10 μm .

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DOR localizes to autophagosomes when autophagy is activated. (A) Confocal images of HeLa cells transiently transfected with DOR and GFP‐LC3, and incubated for 1h with DMEM, HBSS (starvation), 2 μM rapamycin or HBSS containing 50 μM chloroquine. Nuclei were labelled with DAPI. Scale bars, 10 μm.


( B , C ) Confocal images of HeLa cells transiently transfected with DOR or GFP ‐ LC3 and incubated with 50 nM Lysotracker in DMEM or HBSS . Scale bars , 10 μm .

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(B,C) Confocal images of HeLa cells transiently transfected with DOR or GFP‐LC3 and incubated with 50 nM Lysotracker in DMEM or HBSS. Scale bars, 10 μm.


( D ) Confocal images of HeLa cells transiently transfected with DOR and LAMP1 ‐ GFP , or with YFP ‐ LC3 and LAMP1 ‐ GFP . Scale bars , 10 μm . Contrast ‐ corrected merge RGB pictures and the Z ‐ projection of Product of the differences from the mean ( PDM ) images are shown in all panels . Colour scales of PDM images have different maximal values , so the different conditions are not comparable from these projections . Product of the differences from the mean ( PDM ) values closer to 1 show reliable colocalized pixels . DAPI , 4 ′ , 6 ‐ diamidino ‐ 2 ‐ phenylindole ; DMEM , Dulbecco ' s modified Eagle medium ; DOR , diabetes ‐ and obesity ‐ regulated gene ; GFP , green fluorescent protein ; HBSS , Hank ' s balanced salt solution ; LAMP1 , lysosomal ‐ associated membrane protein 1 ; LC3 , microtubule ‐ associated protein 1A / 1B ‐ light chain 3 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 images of HeLa cells transiently transfected with DOR and LAMP1‐GFP, or with YFP‐LC3 and LAMP1‐GFP. Scale bars, 10 μm. Contrast‐corrected merge RGB pictures and the Z‐projection of Product of the differences from the mean (PDM) images are shown in all panels. Colour scales of PDM images have different maximal values, so the different conditions are not comparable from these projections. Product of the differences from the mean (PDM) values closer to 1 show reliable colocalized pixels. DAPI, 4′,6‐diamidino‐2‐phenylindole; DMEM, Dulbecco's modified Eagle medium; DOR, diabetes‐ and obesity‐regulated gene; GFP, green fluorescent protein; HBSS, Hank's balanced salt solution; LAMP1, lysosomal‐associated membrane protein 1; LC3, microtubule‐associated protein 1A/1B‐light chain 3.


( A ) DOR gain ‐ of ‐ function accelerates the degradation of proteins of middle - long half ‐ life . Untreated HeLa cells and those transiently transfected with DOR were incubated in DMEM or HBSS . * Significant effects of starvation , P0 . 01 ; τsignificant effects caused by DOR overexpression , P0 . 01 .

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 O O O O O

(A) DOR gain‐of‐function accelerates the degradation of proteins of middle-long half‐life. Untreated HeLa cells and those transiently transfected with DOR were incubated in DMEM or HBSS. *Significant effects of starvation, P0.01; τsignificant effects caused by DOR overexpression, P0.01.


( B ) DOR overexpression enhances autophagosomal formation . LC3 ‐ GFP ‐ positive vacuoles were counted in 100 transfected HeLa cells ( with GFP ‐ LC3 and TRα1 , or GFP ‐ LC3 and DOR ) . * Significant effects of starvation , P0 . 001 ; τsignificant effects of DOR overexpression , P0 . 001 .

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

(B) DOR overexpression enhances autophagosomal formation. LC3‐GFP‐positive vacuoles were counted in 100 transfected HeLa cells (with GFP‐LC3 and TRα1, or GFP‐LC3 and DOR). *Significant effects of starvation, P0.001; τsignificant effects of DOR overexpression, P0.001.


( C , D ) HeLa cells transiently transfected with empty pCDNA3 or DOR were incubated in DMEM ( basal ) or in HBSS ( starvation ) . Cells were processed by transmission electron microscopy . Arrows indicate autophagosomes . Insets are autophagosomes ( diameter , 407 ± 63 nm ) . Scale bars , 1 μm . Autophagosomes were counted in 50 randomly chosen transfected cells . * Significant effects of starvation , P0 . 05 ; τsignificant effects of DOR overexpression , P0 . 05 . DMEM ; Dulbecco ' s modified Eagle medium ; DOR , diabetes ‐ and obesity ‐ regulated gene ; GFP , green fluorescent protein ; HBSS ; Hank ' s balanced salt solution ; LC3 , microtubule ‐ associated protein 1A / 1B ‐ light chain 3 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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,D) HeLa cells transiently transfected with empty pCDNA3 or DOR were incubated in DMEM (basal) or in HBSS (starvation). Cells were processed by transmission electron microscopy. Arrows indicate autophagosomes. Insets are autophagosomes (diameter, 407±63 nm). Scale bars, 1 μm. Autophagosomes were counted in 50 randomly chosen transfected cells. *Significant effects of starvation, P0.05; τsignificant effects of DOR overexpression, P0.05. DMEM; Dulbecco's modified Eagle medium; DOR, diabetes‐ and obesity‐regulated gene; GFP, green fluorescent protein; HBSS; Hank's balanced salt solution; LC3, microtubule‐associated protein 1A/1B‐light chain 3.


( A , B ) C2C12 myoblasts were previously infected with lentiviruses encoding scrambled RNA , DOR siRNA1 or DOR siRNA2 . Endogenous LC3 was detected by immunofluorescence . LC3 ‐ positive puncta were counted in 100 cells per group under basal conditions or starved for 20 min . Scale bars , 10 μm . * Significant effects of starvation , P0 . 001 ; τsignificant effects caused by DOR knockdown , P0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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,B) C2C12 myoblasts were previously infected with lentiviruses encoding scrambled RNA, DOR siRNA1 or DOR siRNA2. Endogenous LC3 was detected by immunofluorescence. LC3‐positive puncta were counted in 100 cells per group under basal conditions or starved for 20 min. Scale bars, 10 μm. *Significant effects of starvation, P0.001; τsignificant effects caused by DOR knockdown, P0.001.


( C , D ) Scramble or DOR siRNA1 C2C12 cells were processed by transmission electron microscopy . Arrows indicate autophagosomes . Insets are autophagosomes . Scale bars , 1 μm . Autophagosomes were counted in 50 randomly chosen cells . * Significant effects of DOR repression , P0 . 001 .

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

(C,D) Scramble or DOR siRNA1 C2C12 cells were processed by transmission electron microscopy. Arrows indicate autophagosomes. Insets are autophagosomes. Scale bars, 1 μm. Autophagosomes were counted in 50 randomly chosen cells. *Significant effects of DOR repression, P0.001.


( E ) Scramble or DOR siRNA1 C2C12 cells were incubated in DMEM with and without 10 mM 3MA . * Significant effects of DOR knockdown , P0 . 01 ; τsignificant effects caused by 3MA , P0 . 01 . 3MA , 3 ‐ methyladenine ; DMEM , Dulbecco ' s modified Eagle medium ; DOR , diabetes ‐ and obesity ‐ regulated gene ; LC3 , microtubule ‐ associated protein 1A / 1B ‐ light chain 3 .

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

(E) Scramble or DOR siRNA1 C2C12 cells were incubated in DMEM with and without 10 mM 3MA. *Significant effects of DOR knockdown, P0.01; τsignificant effects caused by 3MA, P0.01. 3MA, 3‐methyladenine; DMEM, Dulbecco's modified Eagle medium; DOR, diabetes‐ and obesity‐regulated gene; LC3, microtubule‐associated protein 1A/1B‐light chain 3.


( A ) Decrease of mRNA levels in TubG4 , UAS ‐ CG11347 ‐ RNAi animals compared with TubG4 controls . All measurements were normalized to rp49 . * Significant change , P0 . 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

(A) Decrease of mRNA levels in TubG4, UAS‐CG11347‐RNAi animals compared with TubG4 controls. All measurements were normalized to rp49. *Significant change, P0.05.


( B ) Lysotracker staining of fat bodies . No Lysotracker staining is observed in feeding early third ‐ instar larvae , whereas Lysotracker ‐ positive granules ( red ) accumulate in fat body cells of wandering late third ‐ instar controls ( upper panels , TubG4 / + ) . dDOR knockdown animals ( TubG4 , UAS ‐ CG11347 ‐ RNAi T4 , lower panels ) show decreased staining of Lysotracker ‐ positive granules in wandering late third ‐ instar larvae . Nuclei are shown in blue . Scale bars , 50 μm . ( C ) The number of Lysotracker ‐ positive puncta per unit area are shown for each genotype ( control and RNAi ) , normalized to the value for control cells processed in parallel . * Significant change , P0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Lysotracker staining of fat bodies. No Lysotracker staining is observed in feeding early third‐instar larvae, whereas Lysotracker‐positive granules (red) accumulate in fat body cells of wandering late third‐instar controls (upper panels, TubG4/+). dDOR knockdown animals (TubG4, UAS‐CG11347‐RNAi T4, lower panels) show decreased staining of Lysotracker‐positive granules in wandering late third‐instar larvae. Nuclei are shown in blue. Scale bars, 50 μm. (C) The number of Lysotracker‐positive puncta per unit area are shown for each genotype (control and RNAi), normalized to the value for control cells processed in parallel. *Significant change, P0.05.


( D ) Transmission electron microscopy images of fat body cells from wandering late third ‐ instar larvae . Developmental autophagy results in the accumulation of large autolysosomes ( AL ) in control ( TubG4 / + ) larvae , whereas only a few autolysosomes are seen in CG11347 RNAi fat body cells of the same age . Control and RNAi fat bodies present lipid droplets ( LP ) with a ' striped ' appearance , which is an electron microscopy artefact . Scale bars , 5 nm . dDor , CG11347 , a homologue of DOR ; DOR , diabetes ‐ and obesity ‐ regulated gene ; mRNA , messenger RNA ; wt , wild type .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Transmission electron microscopy images of fat body cells from wandering late third‐instar larvae. Developmental autophagy results in the accumulation of large autolysosomes (AL) in control (TubG4/+) larvae, whereas only a few autolysosomes are seen in CG11347 RNAi fat body cells of the same age. Control and RNAi fat bodies present lipid droplets (LP) with a 'striped' appearance, which is an electron microscopy artefact. Scale bars, 5 nm. dDor, CG11347, a homologue of DOR; DOR, diabetes‐ and obesity‐regulated gene; mRNA, messenger RNA; wt, wild type.


( A , B ) Wild - type ( WT ) ( BY4741 ) cells expressing GFP − Atg8 were grown to mid - log - phase in YPD and were then preincubated with 50 µM cerulenin or 50 µM cerulenin + 0 . 1 mM palmitic / stearic / myristic acids or with DMSO ( - ) in the rich medium for 30 min . Cells were washed and shifted to nitrogen - starvation medium ( SD - N ) for 4 h in the presence of 50 µM cerulenin or 50 µM cerulenin + 0 . 1 mM palmitic / stearic / myristic acids or DMSO . Cells were then lysed and subjected to SDS − PAGE , followed by western blot analysis using anti - GFP and anti - Pgk1 antibodies ( A ) ,

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

(A, B) Wild-type (WT) (BY4741) cells expressing GFP−Atg8 were grown to mid-log-phase in YPD and were then preincubated with 50 µM cerulenin or 50 µM cerulenin + 0.1 mM palmitic/stearic/myristic acids or with DMSO (-) in the rich medium for 30 min. Cells were washed and shifted to nitrogen-starvation medium (SD-N) for 4 h in the presence of 50 µM cerulenin or 50 µM cerulenin + 0.1 mM palmitic/stearic/myristic acids or DMSO. Cells were then lysed and subjected to SDS−PAGE, followed by western blot analysis using anti-GFP and anti-Pgk1 antibodies (A),


( A , B ) Wild - type ( WT ) ( BY4741 ) cells expressing GFP − Atg8were grown to mid - log - phase in YPD and were then preincubated with 50 µM cerulenin or 50 µM cerulenin + 0 . 1 mM palmitic / stearic / myristic acids or with DMSO ( - ) in the rich medium for 30 min . Cells were washed and shifted to nitrogen - starvation medium ( SD - N ) for 4 h in the presence of 50 µM cerulenin or 50 µM cerulenin + 0 . 1 mM palmitic / stearic / myristic acids or DMSO . Cells were then lysed and subjected to SDS − PAGE , followed by western blot analysis using anti - GFP and anti - Pgk1 antibodies ( A ) , or were visualized by fluorescence microscopy ( B )

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

(A, B) Wild-type (WT) (BY4741) cells expressing GFP−Atg8were grown to mid-log-phase in YPD and were then preincubated with 50 µM cerulenin or 50 µM cerulenin + 0.1 mM palmitic/stearic/myristic acids or with DMSO (-) in the rich medium for 30 min. Cells were washed and shifted to nitrogen-starvation medium (SD-N) for 4 h in the presence of 50 µM cerulenin or 50 µM cerulenin + 0.1 mM palmitic/stearic/myristic acids or DMSO. Cells were then lysed and subjected to SDS−PAGE, followed by western blot analysis using anti-GFP and anti-Pgk1 antibodies (A), or were visualized by fluorescence microscopy (B)


( C ) Cells ( TOS038 ) expressing Atg1 - GFP were grown to mid - log phase and were then preincubated in YPD with 50 µM cerulenin or with DMSO for 30 min . Cells were washed , shifted to SD - N for 4 h , and then visualized by fluorescence microscopy . Scale bar , 5 μm .

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

(C) Cells (TOS038) expressing Atg1-GFP were grown to mid-log phase and were then preincubated in YPD with 50 µM cerulenin or with DMSO for 30 min. Cells were washed, shifted to SD-N for 4 h, and then visualized by fluorescence microscopy. Scale bar, 5 μm.


( D ) WT ( BY4741 ) and pep4 ∆ ( TOS015 ) strains were grown to mid - log phase and preincubated in YPD with cerulenin or DMSO for 30 min . Cells were pulse - labeled for 10 min with [ 35S ] methionine and cysteine and chased for the indicated time periods . Cell lysates were subjected to immunoprecipitation with anti - Ape1 antibodies followed by SDS ‒ PAGE and X - ray film to detect radioactive signals . prApe1 , premature Ape1 ; mApe1 , mature Ape1 . (

O O 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

(D) WT (BY4741) and pep4∆ (TOS015) strains were grown to mid-log phase and preincubated in YPD with cerulenin or DMSO for 30 min. Cells were pulse-labeled for 10 min with [35S] methionine and cysteine and chased for the indicated time periods. Cell lysates were subjected to immunoprecipitation with anti-Ape1 antibodies followed by SDS‒PAGE and X-ray film to detect radioactive signals. prApe1, premature Ape1; mApe1, mature Ape1. (


( E ) Cells of the fas1 ∆ ( TOS029 ) strain expressing GFP ‒ Atg8 were grown to mid - log phase in YPD + 0 . 1 mM palmitic / stearic / myristic acids and shifted either to the same medium or to YPD without fatty acids for 30 min . Cells were then shifted to SD - N for the indicated times . Cell lysates were subjected to SDS ‒ PAGE , followed by western blot analysis using anti - GFP antibodies .

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(E) Cells of the fas1∆ (TOS029) strain expressing GFP‒Atg8 were grown to mid-log phase in YPD + 0.1 mM palmitic/stearic/myristic acids and shifted either to the same medium or to YPD without fatty acids for 30 min. Cells were then shifted to SD-N for the indicated times. Cell lysates were subjected to SDS‒PAGE, followed by western blot analysis using anti-GFP antibodies.


( F ) WT ( BY4741 ) cells were grown to mid - log phase and then preincubated in YPD with 25 µM or 50 µM cerulenin ( 25 , 50 ) or with DMSO ( - ) in rich medium for 30 min . Cells were washed and shifted to SD - N in the presence of cerulenin ( 25 , 50 ) or DMSO ( - ) . Cells were lysed and subjected to SDS ‒ PAGE , followed by western blot analysis using anti - GFP and anti - Pgk1 antibodies . cer , cerulenin ; DIC , Differential interference contrast ; FA , fatty acids ; SD - N , nitrogen - starvation medium ; WT , wild - type YPD , complete medium .

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

(F) WT (BY4741) cells were grown to mid-log phase and then preincubated in YPD with 25 µM or 50 µM cerulenin (25, 50) or with DMSO (-) in rich medium for 30 min. Cells were washed and shifted to SD-N in the presence of cerulenin (25, 50) or DMSO (-). Cells were lysed and subjected to SDS‒PAGE, followed by western blot analysis using anti-GFP and anti-Pgk1 antibodies. cer, cerulenin; DIC, Differential interference contrast; FA, fatty acids; SD-N, nitrogen-starvation medium; WT, wild-type YPD, complete medium.


( A ) Cells ( TN124 strain ) were grown to mid - log phase and preincubated in YPD with 50 µM cerulenin for the indicated time periods ( Cer preincubation ) or incubated with DMSO without preincubation ] ( - ) . Cells were then shifted to SD - N for 3 h with the addition of DMSO or 50 µM cerulenin ( cer ) . Autophagic activity was measured by alkaline phosphatase assay . Error bars represent the s . e . m . of three independent experiments . * P < 0 . 05 ( Student ' s t - test ) .

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

(A) Cells (TN124 strain) were grown to mid-log phase and preincubated in YPD with 50 µM cerulenin for the indicated time periods (Cer preincubation) or incubated with DMSO without preincubation] (-). Cells were then shifted to SD-N for 3 h with the addition of DMSO or 50 µM cerulenin (cer). Autophagic activity was measured by alkaline phosphatase assay. Error bars represent the s.e.m. of three independent experiments. *P < 0.05 (Student's t-test).


( B ) Cells were grown as in ( A ) , stained with BODIPY , and visualized by fluorescence microscopy . Scale bar , 5 μm .

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

(B) Cells were grown as in (A), stained with BODIPY, and visualized by fluorescence microscopy. Scale bar, 5 μm.


( C ) WT ( SCY62 ) and tagΔsteΔ ( H1246 ) cells were grown to mid - log phase in YPD and shifted to SD - N for 3 h . Cells were stained with Nile red and visualized by fluorescence microscopy . Scale bar , 5 μm .

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(C) WT (SCY62) and tagΔsteΔ (H1246) cells were grown to mid-log phase in YPD and shifted to SD-N for 3 h. Cells were stained with Nile red and visualized by fluorescence microscopy. Scale bar, 5 μm.


( D ) WT ( SCY62 ) and tag ∆ ste ∆ ( H1246 ) cells expressing GFP − Atg8 were grown to mid - log phase in YPD and shifted to SD - N for the indicated time periods . Cell lysates were subjected to SDS − PAGE , followed by western blot analysis using anti - GFP , anti - Ape1 ( prApe1 , premature Ape1 ; mApe1 , mature Ape1 ) , and anti - Fas1 and anti - Pgk1 antibodies .

O O O O O O O O O O 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

(D) WT (SCY62) and tag∆ste∆ (H1246) cells expressing GFP−Atg8 were grown to mid-log phase in YPD and shifted to SD-N for the indicated time periods. Cell lysates were subjected to SDS−PAGE, followed by western blot analysis using anti-GFP, anti-Ape1 (prApe1, premature Ape1; mApe1, mature Ape1), and anti-Fas1 and anti-Pgk1 antibodies.


( E ) WT ( SCY62 ) and tag ∆ ste ∆ ( H1246 ) cells were grown to mid - log phase in YPD and shifted to SD - N for 2 h . GFP − Atg8 was visualized by fluorescence microscopy . Scale bar , 5 μm . cer , cerulenin ; DIC , Differential interference contrast ; FA , fatty acids ; SD - N , nitrogen - starvation medium ; WT , wild - type , YPD , complete medium .

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

(E) WT (SCY62) and tag∆ste∆ (H1246) cells were grown to mid-log phase in YPD and shifted to SD-N for 2 h. GFP−Atg8 was visualized by fluorescence microscopy. Scale bar, 5 μm. cer, cerulenin; DIC, Differential interference contrast; FA, fatty acids; SD-N, nitrogen-starvation medium; WT, wild-type, YPD, complete medium.


( A ) GAL - DGA1GAL - ARE2lro1Δ are1Δ ( FYS118 strain ) was grown on SC ( synthetic minimal medium without dextrose ) + raffinoseovernight , diluted to OD 0 . 4 , and grown to mid - log phase in either SC + glucose or SC + galactose medium . Cells were shifted to SD - N for 2 h , stained with BODIPY , and visualized by fluorescencemicroscopy . Scale bar , 5 μm .

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

(A) GAL-DGA1GAL-ARE2lro1Δ are1Δ (FYS118 strain) was grown on SC (synthetic minimal medium without dextrose) + raffinoseovernight, diluted to OD 0.4, and grown to mid-log phase in either SC + glucose or SC + galactose medium. Cells were shifted to SD-N for 2 h, stained with BODIPY, and visualized by fluorescencemicroscopy. Scale bar, 5 μm.


( B ) WT ( BY4741 ) and GAL - DGA1 GAL - ARE2 lro1 ∆ are1 ∆ ( FYS118 ) cells expressing GFP − Atg8 were grown as in A . Cells were lysed at the indicated times and subjected to SDS ‒ PAGE , followed by western blot analysis using anti - GFP antibodies . (

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) WT (BY4741) and GAL-DGA1 GAL-ARE2 lro1∆ are1∆ (FYS118) cells expressing GFP−Atg8 were grown as in A. Cells were lysed at the indicated times and subjected to SDS‒PAGE, followed by western blot analysis using anti-GFP antibodies. (


( C , D ) WT ( BY4741 ) and GAL - DGA1 GAL - ARE2 lro1 ∆ are1 ∆ ( FYS118 ) cells expressing GFP − Atg8 ( C ) or GFP − Atg1 ( D ) were grown as in A and visualized by fluorescence microscopy after 2 h in SD - N . Scale bar , 5 μm . DIC , Differential interference contrast ; SD - N , nitrogen - starvation medium ; WT , wild - type .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, D) WT (BY4741) and GAL-DGA1 GAL-ARE2 lro1∆ are1∆ (FYS118) cells expressing GFP−Atg8 (C) or GFP−Atg1 (D) were grown as in A and visualized by fluorescence microscopy after 2 h in SD-N. Scale bar, 5 μm. DIC, Differential interference contrast; SD-N, nitrogen-starvation medium; WT, wild-type.


( A ) WT ( SCY62 ) , tag ∆ ste ∆ ( H1246 ) , tag ∆ ( H1226 ) and ste ∆ ( H1112 ) cells expressing GFP − Atg8 were grown to mid - log phase in YPD and shifted to SD - N for 6 h . Cells were stained with BODIPY and visualized by fluorescence microscopy . Scale bar , 5 μm .

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

(A) WT (SCY62), tag∆ste∆ (H1246), tag∆ (H1226) and ste∆ (H1112) cells expressing GFP−Atg8 were grown to mid-log phase in YPD and shifted to SD-N for 6 h. Cells were stained with BODIPY and visualized by fluorescence microscopy. Scale bar, 5 μm.


( A ) WT ( SCY62 ) , tag ∆ ste ∆ ( H1246 ) , tag ∆ ( H1226 ) and ste ∆ ( H1112 ) cells expressing GFP − Atg8 were grown to mid - log phase in YPD and shifted to SD - N for the indicated time periods . Cells were lysed and subjected to SDS − PAGE , followed by western blot analysis using anti - GFP antibodies . ( B ) Quantification of the GFP / GFP − Atg8 ratio . Error bars represent the s . e . m . of three independent experiments . * P < 0 . 05 ( Student ' s t - test ) . (

(A) WT (SCY62), tag∆ste∆ (H1246), tag∆ (H1226) and ste∆ (H1112) cells expressing GFP−Atg8 were grown to mid-log phase in YPD and shifted to SD-N for the indicated time periods. Cells were lysed and subjected to SDS−PAGE, followed by western blot analysis using anti-GFP antibodies. (B) Quantification of the GFP/GFP−Atg8 ratio. Error bars represent the s.e.m. of three independent experiments. *P < 0.05 (Student's t-test). (


. ( C ) WT ( SCY62 ) , tag ∆ ste ∆ ( H1246 ) , tag ∆ ( H1226 ) and ste ∆ ( H1112 ) cells expressing GFP - Atg8 were grown to mid - log phase in YPD and shifted to SD - N for 2 h . GFP − Atg8 was visualized by fluorescence microscopy . Scale bar , 5 μm . (

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

. (C) WT (SCY62), tag∆ste∆ (H1246), tag∆ (H1226) and ste∆ (H1112) cells expressing GFP-Atg8 were grown to mid-log phase in YPD and shifted to SD-N for 2 h. GFP−Atg8 was visualized by fluorescence microscopy. Scale bar, 5 μm. (


( D ) WT ( SCY62 ) , tag ∆ ste ∆ ( H1246 ) , tag ∆ ( H1226 ) and ste ∆ ( H1112 ) cells were grown as in ( A ) . Lysates were subjected to SDS − PAGE in urea gel , followed by western blot analysis using anti - Atg8 and anti - Pgk1 antibodies . Atg8 I , non - lipidated Atg8 ; Atg8 II , lipidated Atg8 . DIC , Differential interference contrast ; SD - N , nitrogen - starvation medium ; WT , wild type .

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

(D) WT (SCY62), tag∆ste∆ (H1246), tag∆ (H1226) and ste∆ (H1112) cells were grown as in (A). Lysates were subjected to SDS−PAGE in urea gel, followed by western blot analysis using anti-Atg8 and anti-Pgk1 antibodies. Atg8 I, non-lipidated Atg8; Atg8 II, lipidated Atg8. DIC, Differential interference contrast; SD-N, nitrogen-starvation medium; WT, wild type.


Fig . 6 Lack of lipid droplets inhibits starvation - induced formation of autophagosomes . ( A , H ) WT pep4 ∆ ( pep4 ∆ ) ( A , B ) , tag ∆ ste ∆ pep4 ∆ ( tag ∆ ste ∆ pep4 ∆ ) ( C , D ) , ste ∆ pep4 ∆ ( ste ∆ pep4 ∆ ) ( E , F ) and tag ∆ pep4 ∆ ( tag ∆ pep4 ∆ ) ( G , H ) cells were grown to an exponential phase in YPD before being starved in SD - N for 2 h , and were then processed for electron microscopy . H is a magnification of the box in panel G . Magnification of proliferating ER in a tag ∆ ste ∆ ( H1246 ) cell is shown in ( I ) Asterisks indicate autophagic bodies . N , nucleus ; PM , plasma membrane ; CW , cell wall ; ER , endoplasmic reticulum ; M , mitochondrion ; V , vacuole ; # , trehalose ; F , ER proliferation . Scale bars : 1 µm ( A - H ) and 200 nm ( I ) . ( J ) Average number of autophagic bodies per cell section was determined by counting 100 randomly selected cell profiles . Error bars represent standard deviations fromcounting of the three grids . * * * P < 0 . 001 ( Student ' s t - test ) . SD - N , nitrogen - starvation medium ; WT , wild type YPD , complete medium .

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

Fig. 6 Lack of lipid droplets inhibits starvation-induced formation of autophagosomes. (A, H) WT pep4∆ (pep4∆) (A,B), tag∆ste∆ pep4∆ (tag∆ste∆pep4∆)(C,D), ste∆ pep4∆ (ste∆pep4∆)(E,F) and tag∆ pep4∆ (tag∆pep4∆) (G,H) cells were grown to an exponential phase in YPD before being starved in SD-N for 2 h, and were then processed for electron microscopy. H is a magnification of the box in panel G. Magnification of proliferating ER in a tag∆ste∆ (H1246) cell is shown in (I) Asterisks indicate autophagic bodies. N, nucleus; PM, plasma membrane; CW, cell wall; ER, endoplasmic reticulum; M, mitochondrion; V, vacuole; #, trehalose; F, ER proliferation. Scale bars: 1 µm (A-H) and 200 nm (I). (J) Average number of autophagic bodies per cell section was determined by counting 100 randomly selected cell profiles. Error bars represent standard deviations fromcounting of the three grids. ***P < 0.001 (Student's t-test). SD-N,nitrogen-starvation medium; WT, wild type YPD, complete medium.


( A ) WT ( BY4741 ) , tgl1 ∆ , yeh1 ∆ , and yeh2 ∆ cells expressing GFP − Atg8 were grown to mid - log phase in YPD and shifted to SD - N for the indicated time periods . Cells were lysed and subjected to SDS − PAGE , followed by western blot analysis using anti - GFP antibodies . Quantification of the GFP / GFP − Atg8 ratio is presented on the right . Error bars represent the s . e . m . of three independent experiments . * P < 0 . 05 ( Student ' s t - test ) .

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

(A) WT (BY4741), tgl1∆, yeh1∆ ,and yeh2∆ cells expressing GFP−Atg8 were grown to mid-log phase in YPD and shifted to SD-N for the indicated time periods. Cells were lysed and subjected to SDS−PAGE, followed by western blot analysis using anti-GFP antibodies. Quantification of the GFP/GFP−Atg8 ratio is presented on the right. Error bars represent the s.e.m. of three independent experiments. *P < 0.05 (Student's t-test).


( B ) WT ( BY4741 ) and tgl3 ∆ tgl4 ∆ tgl5 ∆ cells expressing GFP − Atg8 were grown , treated , lysed , and western blotted as in ( A ) . Quantification of the GFP / GFP − Atg8 ratio is presented on the right . Error bars represent the s . e . m . of three independent experiments . * P < 0 . 05 ( Student ' s t - test ) . (

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) WT (BY4741) and tgl3∆tgl4∆tgl5∆ cells expressing GFP−Atg8 were grown, treated, lysed, and western blotted as in (A). Quantification of the GFP/GFP−Atg8 ratio is presented on the right. Error bars represent the s.e.m. of three independent experiments. *P < 0.05 (Student's t-test). (


( C ) WT ( BY4741 ) , ayr1 ∆ and ldh1 ∆ cells expressing GFP − Atg8 were grown , treated , lysed , and western blotted as in ( A ) . Quantification of the GFP / GFP − Atg8 ratio is presented on the right . Error bars represent the s . e . m . of three independent experiments . * P < 0 . 05 ( Student ' s t - test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) WT (BY4741), ayr1∆ and ldh1∆ cells expressing GFP−Atg8 were grown, treated, lysed, and western blotted as in (A). Quantification of the GFP/GFP−Atg8 ratio is presented on the right. Error bars represent the s.e.m. of three independent experiments. *P < 0.05 (Student's t-test).


( D ) WT ( BY4741 ) , ldh1 ∆ ayr1 ∆ cells expressing GFP − Atg8 were grown , treated , lysed , and western blotted as in ( A ) . Quantification of the GFP / GFP − Atg8 ratio is presented on the right . Error bars represent the s . e . m . of three independent experiments . * * P < 0 . 01 ( Student ' s - t test ) .

(D) WT (BY4741), ldh1∆ayr1∆ cells expressing GFP−Atg8 were grown, treated, lysed, and western blotted as in (A). Quantification of the GFP/GFP−Atg8 ratio is presented on the right. Error bars represent the s.e.m. of three independent experiments. **P < 0.01 (Student's-t test).


( E ) WT ( BY4741 ) , ice2 ∆ and ldb16 ∆ cells expressing GFP − Atg8 were grown , treated , lysed , and western blotted as in ( A ) . Quantification of the GFP / GFP − Atg8 ratio is presented on the right . Error bars represent the s . e . m . of three independent experiments . * P < 0 . 05 , * * P < 0 . 01 * * * P < 0 . 001 ( Student ' s t - test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) WT (BY4741), ice2∆ and ldb16∆ cells expressing GFP−Atg8 were grown, treated, lysed, and western blotted as in (A). Quantification of the GFP/GFP−Atg8 ratio is presented on the right. Error bars represent the s.e.m. of three independent experiments. *P < 0.05, **P < 0.01 ***P < 0.001 (Student's t-test).


( F ) WT ( BY4741 ) , ice2 ∆ and yeh1 ∆ cells expressing GFP − Atg8 were grown to mid - log phase in YPD and shifted to SD - N for 4 h . GFP − Atg8 was visualized by fluorescence microscopy . Scale bar , 5 μm . DIC , Differential interference contrast ; SD - N , nitrogen - starvation medium ; WT , wild type .

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

(F) WT (BY4741), ice2∆ and yeh1∆ cells expressing GFP−Atg8 were grown to mid-log phase in YPD and shifted to SD-N for 4 h. GFP−Atg8 was visualized by fluorescence microscopy. Scale bar, 5 μm. DIC, Differential interference contrast; SD-N, nitrogen-starvation medium; WT, wild type.


a , Correlative light and electron microscopy of HeLa / GFP - LC3 cell infected with SIN / mCherry . capsid virus . Top left , deconvolved image of red and green fluorescence channels ; middle , three - dimensional surface reconstruction of red and green channels ; right , yellow ( red + green ) colocalization channel . Arrows denote yellow puncta that correspond to ' 1 ' and ' 2 ' in electron microscopy images below . Bottom left , electron microscopy of identical cell ; middle and right , high magnification images of insets ' 1 ' and ' 2 ' . Scale bars , left , 10 µm ; middle and right , 200 nm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Correlative light and electron microscopy of HeLa/GFP-LC3 cell infected with SIN/mCherry.capsid virus. Top left, deconvolved image of red and green fluorescence channels; middle, three-dimensional surface reconstruction of red and green channels; right, yellow (red + green) colocalization channel. Arrows denote yellow puncta that correspond to '1' and '2' in electron microscopy images below. Bottom left, electron microscopy of identical cell; middle and right, high magnification images of insets '1' and '2'. Scale bars, left, 10 µm; middle and right, 200 nm.


a , Electron microscopy analysis of wild - type ( WT ) and Smurf1 - / - ( KO ) MEFs grown in normal media or EBSS ( starvation ) for 4 h . Arrowheads , representative autophagosomes ; arrows , representative autolysosomes . Scale bars , 500 nm .

a, Electron microscopy analysis of wild-type (WT) and Smurf1-/- (KO) MEFs grown in normal media or EBSS (starvation) for 4 h. Arrowheads, representative autophagosomes; arrows, representative autolysosomes. Scale bars, 500 nm.


b , Western blot analyses of LC3 - I / II ( non - lipidated and lipidated forms of MAP1LC3 , respectively ) and p62 levels in MEFs of indicated genotype .

O O O O O O O O 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, Western blot analyses of LC3-I/II (non-lipidated and lipidated forms of MAP1LC3, respectively) and p62 levels in MEFs of indicated genotype.


c , Quantification of colocalization of SIN / mCherry . capsid and GFP - LC3 in indicated MEFs 16 h after SIN / mCherry . capsid / GFP - LC3 infection . Data shown represent mean ± s . e . m . number of colocalized red and green puncta per cell for 50 cells per well in triplicate samples . * P < 0 . 001 against wild - type , Students 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

c, Quantification of colocalization of SIN/mCherry.capsid and GFP-LC3 in indicated MEFs 16 h after SIN/mCherry.capsid/GFP-LC3 infection. Data shown represent mean ± s.e.m. number of colocalized red and green puncta per cell for 50 cells per well in triplicate samples. *P<0.001 against wild-type, Students t-test.


d , Representative confocal micrographs of colocalization of SIN / mCherry . capsid and GFP - LC3 in indicated MEFs 16 h after SIN / mCherry . capsid / GFP - LC3 infection images used for quantification in c . Arrows , colocalized red and green puncta . Scale bar , 15 & amp ; amp ; mgr ; m .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Representative confocal micrographs of colocalization of SIN/mCherry.capsid and GFP-LC3 in indicated MEFs 16 h after SIN/mCherry.capsid/GFP-LC3 infection images used for quantification in c. Arrows, colocalized red and green puncta. Scale bar, 15 &amp;mgr;m.


e , Representative electron microscopy images of indicated MEFs infected with HSV - 1 ( strain 17termA ) . White arrowheads , partially degraded viral nucleocapsids inside autolysosomes ; black arrowheads , intact viral nucleocapsids inside viral vesicles . Scale bar , 0 . 5 µm . For a - e , similar results were obtained in 3 - 5 independent experiments .

e, Representative electron microscopy images of indicated MEFs infected with HSV-1 (strain 17termA). White arrowheads, partially degraded viral nucleocapsids inside autolysosomes; black arrowheads, intact viral nucleocapsids inside viral vesicles. Scale bar, 0.5 µm. For a-e, similar results were obtained in 3-5 independent experiments.


a , Representative mitochondrial morphology in Smurf1 + / + ( wild - type ) and Smurf1 − / − ( KO ) MEFs transfected with indicated construct and treated with DMSO or 10 µM CCCP for 24 h . b , Quantification of percentage of total cells with a diffuse accumulation of abnormal fragmented mitochondria and lack of mitochondrial clearance . Results shown represent combined data from 3 - 5 experiments per condition with triplicate wells ( of at least 100 cells per well ) analysed for each condition per experiment . Shown are mean ± s . e . m . for average values from each experiment . Similar results were observed in each independent experiment . * P 0 . 001 , Students t - test . c , Measurement of mitochondrial fractional area ( percentage of total cellular area ) in MEFs treated as in a . Results shown represent mean ± s . e . m . for 50 cells per condition .

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

a, Representative mitochondrial morphology in Smurf1+/+ (wild-type) and Smurf1−/− (KO) MEFs transfected with indicated construct and treated with DMSO or 10 µM CCCP for 24 h. b, Quantification of percentage of total cells with a diffuse accumulation of abnormal fragmented mitochondria and lack of mitochondrial clearance. Results shown represent combined data from 3-5 experiments per condition with triplicate wells (of at least 100 cells per well) analysed for each condition per experiment. Shown are mean ± s.e.m. for average values from each experiment. Similar results were observed in each independent experiment. *P  0.001, Students t-test. c, Measurement of mitochondrial fractional area (percentage of total cellular area) in MEFs treated as in a. Results shown represent mean ± s.e.m. for 50 cells per condition.


d , Representative confocal micrographs of KO MEFs transfected with YFP - SMURF1 wild - type or YFP - SMURF1Dgr ; C2 ( Dgr ; C2 ) and treated for 4 h with DMSO or CCCP .

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d, Representative confocal micrographs of KO MEFs transfected with YFP-SMURF1 wild-type or YFP-SMURF1Dgr;C2 (Dgr;C2) and treated for 4 h with DMSO or CCCP.


e , Representative confocal micrographs of KO MEFs transfected with GFP - LC3 and wild - type mCherry - SMURF1 ( WT ) or mCherry - SMURF1Dgr ; C2 ( Dgr ; C2 ) and treated for 4 h with CCCP . Inset , upper right , formation of completed autophagosome around a damaged mitochondrion associated with wild - type SMURF1 ; insets , lower right , incomplete autophagosomes or absence of LC3 signal around mitochondria associated with SMURF1Dgr ; C2 . See also Supplementary Figs 10 and 11 for enlarged images .

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e, Representative confocal micrographs of KO MEFs transfected with GFP-LC3 and wild-type mCherry-SMURF1 (WT) or mCherry-SMURF1Dgr;C2 (Dgr;C2) and treated for 4 h with CCCP. Inset, upper right, formation of completed autophagosome around a damaged mitochondrion associated with wild-type SMURF1; insets, lower right, incomplete autophagosomes or absence of LC3 signal around mitochondria associated with SMURF1Dgr;C2. See also Supplementary Figs 10 and 11 for enlarged images.


f , Representative electron microscopy images of indicated tissues from 10 - month - oldSmurf1 + / + ( WT ) or Smurf1 - / - ( KO ) mice . Arrows , representative abnormal mitochondria . Scale bars , 1 µm . Similar abnormalities were observed throughout entire electron microscopy tissue section for each mouse and in tissue samples from three different mice for each genotype .

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f, Representative electron microscopy images of indicated tissues from 10-month-oldSmurf1+/+ (WT) or Smurf1-/- (KO) mice. Arrows, representative abnormal mitochondria. Scale bars, 1 µm. Similar abnormalities were observed throughout entire electron microscopy tissue section for each mouse and in tissue samples from three different mice for each genotype.


G , Benzidine staining of whole spleen [ top ] or marrow [ bottom ] from 3 month old mice [ left ] or 3 year old naked mole - rats [ right ] . Scale bar 250µm , arrows indicate nucleated erythroid progenitors ( NEPs ) .

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G, Benzidine staining of whole spleen [top] or marrow [bottom] from 3 month old mice [left] or 3 year old naked mole-rats [right]. Scale bar 250µm, arrows indicate nucleated erythroid progenitors (NEPs).


H , Relative counts of Benzidine - stained cytospins from whole spleen or WBM . p - value determined by Sidak ' s Two - way ANOVA comparing BM vs spleen between mouse ( n = 5 ) and naked mole - rat ( n = 8 ) . I , Relative counts of Benzidine - stained cytospins from naked mole - rat sorted spleen fractions ; p - value determined by Sidak ' s Two - way ANOVA comparing BM vs spleen ; n = 4 .

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H, Relative counts of Benzidine-stained cytospins from whole spleen or WBM. p-value determined by Sidak's Two-way ANOVA comparing BM vs spleen between mouse (n=5) and naked mole-rat (n=8). I, Relative counts of Benzidine-stained cytospins from naked mole-rat sorted spleen fractions; p-value determined by Sidak's Two-way ANOVA comparing BM vs spleen; n=4.


A , Cross - species integration of bulk RNA - Seq datasets . Naked mole - rat BM populations CP1 - 4 were matched to human and murine counterparts for GSEA with MSigDB hallmark genesets . Primitive stem and progenitor [ left ] , hsa : LT - HSC , LIN - / CD34 + / CD38lo / CD90 + / CD45RA - ( n = 7 ) ; HSPC , LIN - / CD34 + / CD38lo ( n = 11 ) ; MPP , LIN - / CD34 + / CD38lo / CD90 - / CD45RA - ( n = 4 ) ; mmu : LT - HSC , LIN - / Sca - 1 + / Kit + / CD150 + / CD48 - ( n = 25 ) ; MPP , LIN - / Sca - 1 + / Kit + / CD150 - / CD48 + ( n = 6 ) ; hgl : LTC , LIN - / Thy1 . 1int / CD34 + ( n = 5 ) . Lymphomyeloid [ center ] , hsa : LMPP , LIN - / CD34 + / CD38lo / CD123lo / CD45RA + ( n = 9 ) ; CLP , LIN - / CD34 + / CD38hi / CD10 + / CD45RA + ( n = 6 ) ; MLP , LIN - / CD34 + / CD38lo / CD90 - / CD45RA + / CD71 - ( n = 4 ) ; CMP , LIN - / CD34 + / CD38hi / CD123lo / CD45RA - ( n = 26 ) ; GMP , LIN - / CD34 + / CD38lo / CD123lo / CD45RA + ( n = 18 ) ; mmu : LMPP , LIN - / Sca - 1 + / Kit + / Flt3hi ( n = 5 ) ; CMP , LIN - / Sca - 1 - / Kit + / CD16 / 32lo / CD34 + ( n = 8 ) ; GMP , LIN - / Sca - 1 - / Kit + / CD16 / 32hi / CD34 + ( n = 16 ) ; hgl : CP1 , LIN + / Thy1 . 1int / CD34 + ( n = 5 ) . Erythroid [ right ] , hsa : MEP , LIN - / CD34 + / CD38hi / CD123 - / CD45RA - ( n = 23 ) ; MKP , LIN - / CD41a + / CD42b + ( n = 3 ) ; ERY , LIN - / CD34lo / - / CD36 + / GYPA + / CD71 + ( n = 12 ) ; mmu : MEP , LIN - / Sca - 1 - / Kit + / CD16 / 32 - / CD34 - ( n = 15 ) ; hgl : CP3 , LIN - / Thy1 . 1lo / CD34 + ( n = 5 ) ; CP4 , LIN - / Thy1 . 1 - / CD34 + ( n = 5 ) . Within each group , cell types were pooled for each species . Shown are pathways related to proliferation and metabolism normalized enrichment score ; GeneRatio , ( signature ∩ term ) / ( signature ∩ all terms ) ; FDR , false discovery rate .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Cross-species integration of bulk RNA-Seq datasets. Naked mole-rat BM populations CP1-4 were matched to human and murine counterparts for GSEA with MSigDB hallmark genesets. Primitive stem and progenitor [left], hsa: LT-HSC, LIN-/CD34+/CD38lo/CD90+/CD45RA- (n=7); HSPC, LIN-/CD34+/CD38lo (n=11); MPP, LIN-/CD34+/CD38lo/CD90-/CD45RA- (n=4); mmu: LT-HSC, LIN-/Sca-1+/Kit+/CD150+/CD48- (n=25); MPP, LIN-/Sca-1+/Kit+/CD150-/CD48+ (n=6); hgl: LTC, LIN-/Thy1.1int/CD34+ (n=5). Lymphomyeloid [center], hsa: LMPP, LIN-/CD34+/CD38lo/CD123lo/CD45RA+ (n=9); CLP, LIN-/CD34+/CD38hi/CD10+/CD45RA+ (n=6); MLP, LIN-/CD34+/CD38lo/CD90-/CD45RA+/CD71- (n=4); CMP, LIN-/CD34+/CD38hi/CD123lo/CD45RA- (n=26); GMP, LIN-/CD34+/CD38lo/CD123lo/CD45RA+ (n=18); mmu: LMPP, LIN-/Sca-1+/Kit+/Flt3hi (n=5); CMP, LIN-/Sca-1-/Kit+/CD16/32lo/CD34+ (n=8); GMP, LIN-/Sca-1-/Kit+/CD16/32hi/CD34+ (n=16); hgl: CP1, LIN+/Thy1.1int/CD34+ (n=5). Erythroid [right], hsa: MEP, LIN-/CD34+/CD38hi/CD123-/CD45RA- (n=23); MKP, LIN-/CD41a+/CD42b+ (n=3); ERY, LIN-/CD34lo/-/CD36+/GYPA+/CD71+ (n=12); mmu: MEP, LIN-/Sca-1-/Kit+/CD16/32-/CD34- (n=15); hgl: CP3, LIN-/Thy1.1lo/CD34+ (n=5); CP4, LIN-/Thy1.1-/CD34+ (n=5). Within each group, cell types were pooled for each species. Shown are pathways related to proliferation and metabolism normalized enrichment score; GeneRatio, (signature ∩ term) / (signature ∩ all terms); FDR, false discovery rate.


B , Seahorse assay with cell types sorted according to ( A ) ; hOPP , human oligopotent progenitors LIN - / CD34 + / CD38 + ; mOPP , LIN - / Sca - 1 - / Kit + . ECAR , extracellular acidification rate ; OCR , oxygen consumption rate . Error bars , s . e . m . ; Human cell types , n = 4 ; mouse , n = 3 , naked mole - rat , n = 3 .

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B, Seahorse assay with cell types sorted according to (A); hOPP, human oligopotent progenitors LIN-/CD34+/CD38+; mOPP, LIN-/Sca-1-/Kit+. ECAR, extracellular acidification rate; OCR, oxygen consumption rate. Error bars, s.e.m.; Human cell types, n=4; mouse, n=3, naked mole-rat, n=3.


C , Mitotracker Red staining in mouse ( n = 4 ) , human ( n = 4 ) and naked mole - rat ( n = 5 ) BM , histogram of merged per - species data . unstained , BM from mouse [ solid ] , human [ dotted ] or NMR [ area under curve ( AUC ) ] ; yLSK , LIN - / Sca - 1 + / Kit + , 3 month old ; oLSK , 24 month ; yLTC , LIN - / Thy1 . 1int / CD34 + , 3 year old ; hHSC , human CD34 + / CD38lo ;

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Mitotracker Red staining in mouse (n=4), human (n=4) and naked mole-rat (n=5) BM, histogram of merged per-species data. unstained, BM from mouse [solid], human [dotted] or NMR [area under curve (AUC)]; yLSK, LIN-/Sca-1+/Kit+, 3 month old; oLSK, 24 month; yLTC, LIN-/Thy1.1int/CD34+, 3 year old; hHSC, human CD34+/CD38lo;


F , Confocal fluorescence imaging of sorted mouse LT - HSCs ( LIN - / Sca - 1 + / Kit + / CD150 + / CD48 - ) and NMR LTCs across age ; Y , young ( mouse , 4 month - old ; NMR , 3 year - old ) ; O , old ( mouse , 24 month - old ) ; MA , middle - aged ( NMR , 11 year - old ) . Scale bar 10µm .

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F, Confocal fluorescence imaging of sorted mouse LT-HSCs (LIN-/Sca-1+/Kit+/CD150+/CD48-) and NMR LTCs across age; Y, young (mouse, 4 month-old; NMR, 3 year-old); O, old (mouse, 24 month-old); MA, middle-aged (NMR, 11 year-old). Scale bar 10µm.


G , H Polarization quantification for G , mouse LT - HSC ( biological n = 3 ) ; and H , NMR LTC ( Y , n = 5 ; MA , n = 4 ) . Line at mean ; error bars , s . e . m . ; p - value determined by Sidak ' s Two - way ANOVA .

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G, H Polarization quantification for G, mouse LT-HSC (biological n=3); and H, NMR LTC (Y, n=5; MA, n=4). Line at mean; error bars, s.e.m.; p-value determined by Sidak's Two-way ANOVA.


( A ) WT and atg1Δ cells grown in YEPD medium were transferred to SD - N or SD - N + 50 mM MES - KOH ( pH 6 . 2 ) medium for 120 hours , and dead cells stained by phloxine B were observed by fluorescent microscopy . Scale bar , 25 µm .

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(A) WT and atg1Δ cells grown in YEPD medium were transferred to SD-N or SD-N +50 mM MES-KOH (pH 6.2) medium for 120 hours, and dead cells stained by phloxine B were observed by fluorescent microscopy. Scale bar, 25 µm.


( B - C ) WT and atg1Δ cells grown nitrogen starved as ( A ) for the indicated times . Cell viability ( B ) and medium pH ( C ) were examined by phloxine B staining and pH meter , respectively . These data represent the average of three independent experiments and bars indicate standard deviations .

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(B-C) WT and atg1Δ cells grown nitrogen starved as (A) for the indicated times. Cell viability (B) and medium pH (C) were examined by phloxine B staining and pH meter, respectively. These data represent the average of three independent experiments and bars indicate standard deviations.


( A ) WT and atg1Δ cells grown in YEPD medium were transferred to SD - N + 50 mM MES - KOH ( pH 6 . 2 ) . After 120 hours , cultures were diluted 5 . 0 × 105 fold and plated onto YEPD agar . The plates were incubated at 30 ° C for four days . Insets indicate the plate overlaid with TTC agar to examine the respiratory competency of formed colonies .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) WT and atg1Δ cells grown in YEPD medium were transferred to SD-N +50 mM MES-KOH (pH 6.2). After 120 hours, cultures were diluted 5.0×105 fold and plated onto YEPD agar. The plates were incubated at 30°C for four days. Insets indicate the plate overlaid with TTC agar to examine the respiratory competency of formed colonies.


( B ) WT , and atg1Δ cells were nitrogen - starved as ( A ) . Cell viability and respiratory competency was determined by phloxine B staining and TTC overlay technique as ( A ) , respectively . The black and gray areas indicate the percentage of viable cells that are respiratory competent or respiratory deficient , respectively .

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(B) WT, and atg1Δ cells were nitrogen-starved as (A). Cell viability and respiratory competency was determined by phloxine B staining and TTC overlay technique as (A), respectively. The black and gray areas indicate the percentage of viable cells that are respiratory competent or respiratory deficient, respectively.


( C ) WT , atg1Δ , atg2Δ , atg7Δ , atg11Δ , atg15Δ , and atg32Δ cells were nitrogen - starved as ( A ) . Cell viability and respiratory competency was determined by phloxine B staining and TTC overlay technique as ( A ) , respectively . The black and gray areas indicate the percentage of viable cells that are respiratory competent or respiratory deficient , respectively . These data represent the average of three independent experiments and bars indicate standard deviations .

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(C) WT, atg1Δ, atg2Δ, atg7Δ, atg11Δ, atg15Δ, and atg32Δ cells were nitrogen-starved as (A). Cell viability and respiratory competency was determined by phloxine B staining and TTC overlay technique as (A), respectively. The black and gray areas indicate the percentage of viable cells that are respiratory competent or respiratory deficient, respectively. These data represent the average of three independent experiments and bars indicate standard deviations.


( A ) WT and atg1Δ cells expressing mitochondria targeted mCherry cultured in SD - N + 50 mM MES - KOH ( pH 6 . 2 ) medium for 120 hours were observed by fluorescent microscopy . Mitochondrial DNA was stained with SYBR green I , and mitochondria were visualized by mitochondria targeted mCherry . Scale bar , 2 µm .

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(A) WT and atg1Δ cells expressing mitochondria targeted mCherry cultured in SD-N +50 mM MES-KOH (pH 6.2) medium for 120 hours were observed by fluorescent microscopy. Mitochondrial DNA was stained with SYBR green I, and mitochondria were visualized by mitochondria targeted mCherry. Scale bar, 2 µm.


( B - C ) WT and atg1Δ cells were transferred to SD - N + 50 mM MES - KOH ( pH 6 . 2 ) medium for the indicated time . ROS accumulation was detected by DHE staining ( B ) . Each photo contains about 200 cells . Scale bar , 20 µm . ( C ) shows quantification of ROS accumulated cells ( n > 200 cells ) .

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(B-C) WT and atg1Δ cells were transferred to SD-N +50 mM MES-KOH (pH 6.2) medium for the indicated time. ROS accumulation was detected by DHE staining (B). Each photo contains about 200 cells. Scale bar, 20 µm. (C) shows quantification of ROS accumulated cells (n>200 cells).


( D ) WT and atg1Δ cells were transferred to SD - N + 50 mM MES - KOH ( pH 6 . 2 ) with 10 mM NAC for the indicated time . Cell viability was determined by phloxine B staining . Cells from these cultures were plated on YEPD agar and overlaid with TTC agar to examine respiratory competency . The black and gray areas indicate the percentage of viable cells that are respiratory competent or respiratory deficient , respectively . These data represent the average of three independent experiments and bars indicate standard

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(D) WT and atg1Δ cells were transferred to SD-N +50 mM MES-KOH (pH 6.2) with 10 mM NAC for the indicated time. Cell viability was determined by phloxine B staining. Cells from these cultures were plated on YEPD agar and overlaid with TTC agar to examine respiratory competency. The black and gray areas indicate the percentage of viable cells that are respiratory competent or respiratory deficient, respectively. These data represent the average of three independent experiments and bars indicate standard deviations


( A ) WT and atg1Δ cells were transferred to SD - N + 50 mM MES - KOH ( pH 6 . 2 ) medium for the indicated time . Lysates were prepared using the alkaline - trichloroacetic acid method and subjected to immunoprecipitation with anti - Cox2 , anti - Cox4 , anti - Tim17 and anti - Pgk1 . Pgk1 was used as loading control .

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(A) WT and atg1Δ cells were transferred to SD-N +50 mM MES-KOH (pH 6.2) medium for the indicated time. Lysates were prepared using the alkaline-trichloroacetic acid method and subjected to immunoprecipitation with anti-Cox2, anti-Cox4, anti-Tim17 and anti-Pgk1. Pgk1 was used as loading control.


( B ) WT and atg1Δ cells expressing Cta1 - 3 × FLAG or Ctt1 - 3 × FLAG were nitrogen - starved as in ( A ) for the indicated time . Cell lysates were prepared as ( A ) and subjected to immune precipitation with anti - FLAG and anti - Pgk1 . Pgk1 was used as loading control .

(B) WT and atg1Δ cells expressing Cta1-3×FLAG or Ctt1-3×FLAG were nitrogen-starved as in (A) for the indicated time. Cell lysates were prepared as (A) and subjected to immune precipitation with anti-FLAG and anti-Pgk1. Pgk1 was used as loading control.


( A ) WT and atg1Δ cells were transferred to non - buffered SD - N medium for the indicated time . Cell viability was determined by phloxine B staining . Nitrogen - starved cells were plated onto YEPD agar and overlaid with TTC agar to examine the respiratory competency of formed colonies . The black and gray areas indicate the percentage of viable cells that are respiratory competent or respiratory deficient , respectively .

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(A) WT and atg1Δ cells were transferred to non-buffered SD-N medium for the indicated time. Cell viability was determined by phloxine B staining. Nitrogen-starved cells were plated onto YEPD agar and overlaid with TTC agar to examine the respiratory competency of formed colonies. The black and gray areas indicate the percentage of viable cells that are respiratory competent or respiratory deficient, respectively.


( B ) WT , atg1Δ , rho0 , and rho0 atg1Δ cells grown in YEPD medium were transferred to SD - N with or without 10 mM NAC for the indicated time . In the presence of NAC , medium pH was adjusted by using KOH . Cell viability and medium pH were examined by phloxine B staining and pH meter , respectively . These data represent the average of three independent experiments and bars indicate standard deviations .

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(B) WT, atg1Δ, rho0, and rho0 atg1Δ cells grown in YEPD medium were transferred to SD-N with or without 10 mM NAC for the indicated time. In the presence of NAC, medium pH was adjusted by using KOH. Cell viability and medium pH were examined by phloxine B staining and pH meter, respectively. These data represent the average of three independent experiments and bars indicate standard deviations.


B ) Western blot assays for the expression level of TFAM in ten HCC cell lines , one normal liver cell line ( LO2 ) and two non - tumor liver tissues . The protein level was quantified as ratios between TFAM and GAPDH ( n = 3 independent experiments ) . Data information : Graphs show mean ± s . e . m . , two - tailed unpaired t - test . * * P < 0 . 01 .

B) Western blot assays for the expression level of TFAM in ten HCC cell lines, one normal liver cell line (LO2) and two non-tumor liver tissues. The protein level was quantified as ratios between TFAM and GAPDH (n = 3 independent experiments). Data information: Graphs show mean ± s.e.m., two-tailed unpaired t-test. **P < 0.01.


( C ) Western blot and quantitative analysis for the expression of TFAM in HCC cells with TFAM stable knockdown and control cells ( n = 3 independent experiments ) . shCtrl , control shRNA ; shTFAM , shRNA against TFAM ; EV , empty vector ; TFAM , expression vector encoding TFAM . Data information : Graphs show mean ± s . e . m . , two - tailed unpaired t - test . * * P < 0 . 01 .

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(C) Western blot and quantitative analysis for the expression of TFAM in HCC cells with TFAM stable knockdown and control cells (n = 3 independent experiments). shCtrl, control shRNA; shTFAM, shRNA against TFAM; EV, empty vector; TFAM, expression vector encoding TFAM. Data information: Graphs show mean ± s.e.m., two-tailed unpaired t-test. **P < 0.01.


( D , E ) Wound - healing migration assay ( D ) and Transwell migration and invasion assays ( E ) for HCC cells with treatment as indicated ( n = 3 independent experiments ) . Scale bars , 100μm . Data information : Graphs show mean ± s . e . m . , two - tailed unpaired t - test . * * P < 0 . 01 .

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(D, E) Wound-healing migration assay (D) and Transwell migration and invasion assays (E) for HCC cells with treatment as indicated (n = 3 independent experiments). Scale bars, 100μm. Data information: Graphs show mean ± s.e.m., two-tailed unpaired t-test. **P < 0.01.


( G ) Representative H & E images of lung metastasis in mice used in ( F ) . Scale bars , 100μm . ( H , Number of metastatic nodules per lung ( H ) in mice used in ( F ) . Data information : Graphs show mean ± s . e . m . , two - tailed unpaired t - test . * * P < 0 . 01 .

(G) Representative H&E images of lung metastasis in mice used in (F). Scale bars, 100μm. (H, Number of metastatic nodules per lung (H) in mice used in (F). Data information: Graphs show mean ± s.e.m., two-tailed unpaired t-test. **P < 0.01.


( C ) Immunofluorescence analysis of TFAM and Phalloidin staining in human HCC cells from paired clinical samples ( n = 100 ) . The 3D reconstruction of z - stacks from nuclear region is showed in the right side . Red : LifeAct , Blue : DAPI , Green : TFAM . Scale bars , 20μm for microscopy images , 40μm for 3D reconstruction .

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(C) Immunofluorescence analysis of TFAM and Phalloidin staining in human HCC cells from paired clinical samples (n=100). The 3D reconstruction of z-stacks from nuclear region is showed in the right side. Red: LifeAct, Blue: DAPI, Green: TFAM. Scale bars, 20μm for microscopy images, 40μm for 3D reconstruction.


( D ) Statistical analysis for fluorescent intensity of nuclear F - actin in ( A ) , ( B ) and ( C ) . Fluorescence intensity are quantified from at least 30 cells for each group in ( A ) and ( B ) . One - way ANOVA . * P < 0 . 05 ; * * P < 0 . 01 .

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(D) Statistical analysis for fluorescent intensity of nuclear F-actin in (A), (B) and (C). Fluorescence intensity are quantified from at least 30 cells for each group in (A) and (B). One-way ANOVA. *P < 0.05; **P < 0.01.


( E ) Representative H & E images of lung metastasis in mice used in ( D ) . Scale bars , 100μm . ( F and G ) Number of metastatic nodules per lung in mice used in ( F ) and the incidence of lung metastasis ( G ) in mice used in ( D ) . Data information : Graphs show mean ± s . e . m . , two - tailed unpaired t - test . * * P < 0 . 01 .

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(E) Representative H&E images of lung metastasis in mice used in (D). Scale bars, 100μm. (F and G) Number of metastatic nodules per lung in mice used in (F) and the incidence of lung metastasis (G) in mice used in (D). Data information: Graphs show mean ± s.e.m., two-tailed unpaired t-test. **P < 0.01.


F ) Western blot assays for protein expression levels of metastasis related gene in HCC cells ( n = 3 independent experiments ) and tumor tissues from mice ( n = 4 per group ) with treatments as indicated . Data information : Graphs show mean ± s . e . m . , two - tailed unpaired t - test . * P < 0 . 05 ; * * P < 0 . 01 .

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F) Western blot assays for protein expression levels of metastasis related gene in HCC cells (n = 3 independent experiments) and tumor tissues from mice (n =4 per group) with treatments as indicated. Data information: Graphs show mean ± s.e.m., two-tailed unpaired t-test. *P < 0.05; **P < 0.01.


( H Western blot assays levels of FN1 , ITGB3 in HCC cells with treatment as indicated ( n = 3 independent experiments ) . Data information : Graphs show mean ± s . e . m . , two - tailed unpaired t - test . * P < 0 . 05 ; * * P < 0 . 01 .

(H Western blot assays levels of FN1, ITGB3 in HCC cells with treatment as indicated (n = 3 independent experiments). Data information: Graphs show mean ± s.e.m., two-tailed unpaired t-test. *P < 0.05; **P < 0.01.


( A ) Western blot analyses for the protein level of key regulators of actin polymerization in the cytoplasm and nucleus of HCC cells with treatment as indicated . Tublin and Lamin B1 were used as loading controls for the cytoplasm and nucleus , respectively ( n = 3 independent experiments ) .

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(A) Western blot analyses for the protein level of key regulators of actin polymerization in the cytoplasm and nucleus of HCC cells with treatment as indicated. Tublin and Lamin B1 were used as loading controls for the cytoplasm and nucleus, respectively (n = 3 independent experiments).


( B ) Western blot analyses for the protein level in tissues of mouse liver tumor induced by Akt / β - catenin ( n = 3 independent experiments ) .

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(B) Western blot analyses for the protein level in tissues of mouse liver tumor induced by Akt/β-catenin (n = 3 independent experiments).


Representative IHC images of mDia2 nuclear translocation in human ( E ) HCC tissues . NT , non - tumor liver tissues , n = 100 ; MT , metastatic tumor tissues , n = 58 ; NMT , non - metastatic tumor tissues n = 42 . Scale bars , 25μm . ( F ) Positive rates of mDia2 in nucleus and cytoplasm was calculated based on the IHC assay in ( E ) . Graphs show mean ± s . e . m . One - way ANOVA . * * P < 0 . 01 .

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Representative IHC images of mDia2 nuclear translocation in human (E) HCC tissues. NT, non-tumor liver tissues, n=100; MT, metastatic tumor tissues, n=58; NMT, non-metastatic tumor tissues n=42. Scale bars, 25μm. (F) Positive rates of mDia2 in nucleus and cytoplasm was calculated based on the IHC assay in (E). Graphs show mean ± s.e.m. One-way ANOVA. **P < 0.01.


( H and I ) Spearman correlation analysis between the positive rates of nuclear mDia2 and the TFAM expression level ( or positive rates of nuclear F - actin ) based on the IHC assay in ( E ) .

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(H and I) Spearman correlation analysis between the positive rates of nuclear mDia2 and the TFAM expression level (or positive rates of nuclear F-actin) based on the IHC assay in (E).


( D ) Western blot analysis of anti - mDia2 IPs and whole - cell lysates ( WCL ) derived from HCC cells and mouse tumor tissues ( n = 3 independent experiments ) . NT , non - tumor liver tissues ; MT , metastatic tumor tissues ; NMT , non - metastatic tumor tissues .

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(D) Western blot analysis of anti-mDia2 IPs and whole-cell lysates (WCL) derived from HCC cells and mouse tumor tissues (n = 3 independent experiments). NT, non-tumor liver tissues; MT, metastatic tumor tissues; NMT, non-metastatic tumor tissues.


( E ) The level of malonylated mDia2 . Equal amount of immunoprecipitated mDia2 either from TFAM knockdown or control cells was loaded in Western blot assay .

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(E) The level of malonylated mDia2. Equal amount of immunoprecipitated mDia2 either from TFAM knockdown or control cells was loaded in Western blot assay.


( F ) Western blot analysis for the distribution of mDia2 in SNU - 368 cells incubated with malonyl - CoA ( 5 μM ) or ND - 630 ( 10 μM ) ( n = 3 independent experiments ) .

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(F) Western blot analysis for the distribution of mDia2 in SNU-368 cells incubated with malonyl-CoA (5 μM) or ND-630 (10 μM) (n = 3 independent experiments).


( H ) Representative H & E images of lung metastasis in mice used in ( G ) . Scale bars , 100μm . ( I ) Number of metastatic nodules per lung in mice used in ( G ) . Data information : Graphs show mean ± s . e . m . , two - tailed unpaired t - test for , I ) . * P < 0 . 05 ; * * P < 0 . 01 .

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(H) Representative H&E images of lung metastasis in mice used in (G). Scale bars, 100μm. (I) Number of metastatic nodules per lung in mice used in (G). Data information: Graphs show mean ± s.e.m., two-tailed unpaired t-test for , I). *P < 0.05; **P < 0.01.


( A ) IHC scores of TFAM in 429 paired tissues from HCC patients . The relative expression ratio of tumor to non - tumor was log2 - transformed . T , tumor tissues ; NT , non - tumor liver tissues .

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(A) IHC scores of TFAM in 429 paired tissues from HCC patients. The relative expression ratio of tumor to non-tumor was log2-transformed. T, tumor tissues; NT, non-tumor liver tissues.


( B ) mRNA level of TFAM in 50 HCC patients ' paired tissues from TCGA database . The relative expression ratio of tumor to non - tumor was log2 - transformed .

(B) mRNA level of TFAM in 50 HCC patients' paired tissues from TCGA database. The relative expression ratio of tumor to non-tumor was log2-transformed.


( C ) Representative images of IHC staining of TFAM ( top panel ) and scores ( bottom panel ) in HCC tumor tissues . One - way ANOVA . * P < 0 . 05 ; * * P < 0 . 01 . MT , metastatic tumor tissues ; NMT , non - metastatic tumor tissues . Scale bars , 25μm . Boxes : first quartile to third quartile ; Whiskers : minimum value to maximum value ; Central band : median .

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(C) Representative images of IHC staining of TFAM (top panel) and scores (bottom panel) in HCC tumor tissues. One-way ANOVA. *P < 0.05; **P < 0.01. MT, metastatic tumor tissues; NMT, non-metastatic tumor tissues. Scale bars, 25μm. Boxes: first quartile to third quartile; Whiskers: minimum value to maximum value; Central band: median.


( D Kaplan - Meier curves for overall survival of HCC patients stratified by TFAM expression . ( F Kaplan - Meier curves for overall survival of HCC patients stratified by nuclear F - actin positive rates .

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(D Kaplan-Meier curves for overall survival of HCC patients stratified by TFAM expression. (F Kaplan-Meier curves for overall survival of HCC patients stratified by nuclear F-actin positive rates.


E ) the recurrence of HCC patients stratified by TFAM expression . G ) the recurrence of HCC patients stratified by nuclear F - actin positive rates .

E) the recurrence of HCC patients stratified by TFAM expression. G) the recurrence of HCC patients stratified by nuclear F-actin positive rates.


( H ) Representative images of IHC staining of TFAM , FN1 and ITGB3 from two HCC patients . Scale bars , 25μm . ( I ) Spearman correlation analysis between the levels of TFAM and FN1 , IL - 6 , TGFB1 or ITGB3 .

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(H) Representative images of IHC staining of TFAM, FN1 and ITGB3 from two HCC patients. Scale bars, 25μm. (I) Spearman correlation analysis between the levels of TFAM and FN1, IL-6, TGFB1 or ITGB3.


A ‐ F ′ Confocal fluorescent images of wild ‐ type ( A , C , E ) and mtm ‐ 3 ( tm4475 ) ( B , D , F ) embryos at the 1 . 5 ‐ fold stage stained by DAPI ( A - F ) and anti ‐ SQST ‐ 1 ( A ′ , B ′ ) , anti ‐ SEPA ‐ 1 ( C ′ , D ′ ) or anti ‐ LGG ‐ 1 ( E ′ , F ′ ) antibodies . Scale bars : 5 μm .

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A‐F′ Confocal fluorescent images of wild‐type (A, C, E) and mtm‐3(tm4475) (B, D, F) embryos at the 1.5‐fold stage stained by DAPI (A-F) and anti‐SQST‐1 (A′, B′), anti‐SEPA‐1 (C′, D′) or anti‐LGG‐1 (E′, F′) antibodies. Scale bars: 5 μm.


G Western blot analysis of LGG ‐ 1 ‐ I and LGG ‐ 1 - II ( lipidated ) in wild type , epg ‐ 6 , and mtm ‐ 3 .

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G Western blot analysis of LGG‐1‐I and LGG‐1-II (lipidated) in wild type, epg‐6, and mtm‐3.


H mtm ‐ 3 and epg ‐ 6 mutant L1 larvae have shortened mean and maximum life span in the absence of food . At least 300 animals were scored each day .

H mtm‐3 and epg‐6 mutant L1 larvae have shortened mean and maximum life span in the absence of food. At least 300 animals were scored each day.


A - C Fluorescent images of wild ‐ type embryos ( 1 . 5 ‐ fold stage ) expressing GFP : : MTM ‐ 3 ( A ) , GFP : : MTM ‐ 3 ( C459S ) ( B ) , or GFP : : MTM ‐ 3 ( C459S , del PH ‐ G ) ( C ) .

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A-C Fluorescent images of wild‐type embryos (1.5‐fold stage) expressing GFP::MTM‐3 (A), GFP::MTM‐3(C459S) (B), or GFP::MTM‐3(C459S, del PH‐G) (C).


D ‐ G ″ Confocal fluorescent images of wild ‐ type ( D ‐ D ″ ) , mtm ‐ 3 ( E ‐ E ″ ) , atg ‐ 2 ( F ‐ F ″ ) , and vps ‐ 34 ( G ‐ G ″ ) embryos at the 200 ‐ cell stage expressing GFP : : MTM ‐ 3 ( C459S ) and stained by anti ‐ LGG ‐ 1 antibody . Insets show magnified views . Arrows indicate overlapping GFP and LGG ‐ 1 puncta . Scale bars : 5 μm .

D‐G″ Confocal fluorescent images of wild‐type (D‐D″), mtm‐3 (E‐E″), atg‐2 (F‐F″), and vps‐34 (G‐G″) embryos at the 200‐cell stage expressing GFP::MTM‐3(C459S) and stained by anti‐LGG‐1 antibody. Insets show magnified views. Arrows indicate overlapping GFP and LGG‐1 puncta. Scale bars: 5 μm.


H , I Number of GFP : : MTM ‐ 3 ( C459S ) ‐ positive puncta and co ‐ localization of GFP : : MTM ‐ 3 ( C459S ) ‐ and LGG ‐ 1 ‐ positive puncta in various strains [ mtm ‐ 3 ( tm4475 ) , atg ‐ 2 ( bp576 ) , lgg ‐ 1 ( bp500 ) , vps ‐ 34 ( h797 ) ] . At least 10 ( I ) and 15 ( H ) embryos were scored per strain . Data are shown as mean ± SD . Data from different mutant backgrounds were compared with wild type . * * P 0 . 0001 ; N . S . : not statistically different .

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H, I Number of GFP::MTM‐3(C459S)‐positive puncta and co‐localization of GFP::MTM‐3(C459S)‐ and LGG‐1‐positive puncta in various strains [mtm‐3(tm4475), atg‐2(bp576), lgg‐1(bp500), vps‐34(h797)]. At least 10 (I) and 15 (H) embryos were scored per strain. Data are shown as mean ± SD. Data from different mutant backgrounds were compared with wild type. **P 0.0001; N.S.: not statistically different.


J MTM ‐ 3 but not MTM ‐ 3 ( C459S ) exhibits phosphatase activity toward PtdIns3P and PtdIns ( 3 , 5 ) P2 in vitro .

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J MTM‐3 but not MTM‐3(C459S) exhibits phosphatase activity toward PtdIns3P and PtdIns(3,5)P2 in vitro.


A - K Confocal fluorescent images of 200 ‐ cell ‐ stage embryos from the indicated strains stained by both anti ‐ SEPA ‐ 1 and anti ‐ LGG ‐ 1 antibodies . Insets show magnified views . Arrows indicate overlapping SEPA ‐ 1 and LGG ‐ 1 puncta . The wild type contains no visible SEPA ‐ 1 aggregates at this stage and is therefore not shown . Scale bars : 5 μm .

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A-K Confocal fluorescent images of 200‐cell‐stage embryos from the indicated strains stained by both anti‐SEPA‐1 and anti‐LGG‐1 antibodies. Insets show magnified views. Arrows indicate overlapping SEPA‐1 and LGG‐1 puncta. The wild type contains no visible SEPA‐1 aggregates at this stage and is therefore not shown. Scale bars: 5 μm.


L The percentage of SEPA ‐ 1 puncta co ‐ localizing with LGG ‐ 1 in each strain . At least 10 embryos were scored per strain . Data are shown as mean ± SD . * * P 0 . 0001 ; N . S . : not statistically different .

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L The percentage of SEPA‐1 puncta co‐localizing with LGG‐1 in each strain. At least 10 embryos were scored per strain. Data are shown as mean ± SD. **P 0.0001; N.S.: not statistically different.


A - G Confocal fluorescent images of 1 . 5 ‐ fold ‐ stage embryos from the indicated strains expressing both SQST ‐ 1 : : GFP and NUC ‐ 1 : : mCHERRY . Insets show magnified views . Arrows indicate overlapping or closely associated GFP and mCHERRY . Scale bars : 5 μm . The wild type contains no visible SQST ‐ 1 : : GFP puncta at this stage and is therefore not shown .

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A-G Confocal fluorescent images of 1.5‐fold‐stage embryos from the indicated strains expressing both SQST‐1::GFP and NUC‐1::mCHERRY. Insets show magnified views. Arrows indicate overlapping or closely associated GFP and mCHERRY. Scale bars: 5 μm. The wild type contains no visible SQST‐1::GFP puncta at this stage and is therefore not shown.


H , I The percentage of SQST ‐ 1 : : GFP ‐ positive puncta that overlap with ( H ) or are close to ( I ) NUC ‐ 1 : : mCHERRY ‐ positive structures in each strain . At least 10 embryos were scored per strain . Data are shown as mean ± SD . * * P 0 . 0001 ; N . S . : not statistically different .

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H, I The percentage of SQST‐1::GFP‐positive puncta that overlap with (H) or are close to (I) NUC‐1::mCHERRY‐positive structures in each strain. At least 10 embryos were scored per strain. Data are shown as mean ± SD. **P 0.0001; N.S.: not statistically different.


J - L TEM analysis of embryos in wild type ( I ) and mtm ‐ 3 ( tm4475 ) ( J , K ) . Double ‐ membrane autophagosomes ( arrows ) were observed in mtm ‐ 3 but not wild ‐ type embryos . Scale bars : 500 nm .

J-L TEM analysis of embryos in wild type (I) and mtm‐3(tm4475) (J, K). Double‐membrane autophagosomes (arrows) were observed in mtm‐3 but not wild‐type embryos. Scale bars: 500 nm.


A - H Fluorescent images of embryos at the 4 ‐ fold stage in the indicated strains expressing ATG ‐ 18 : : GFP ( A - D ) , ATG ‐ 18 ( FKKG ) : : GFP ( E , F ) , or ATG ‐ 18 ( FTTG ) : : GFP ( G , H ) .

A-H Fluorescent images of embryos at the 4‐fold stage in the indicated strains expressing ATG‐18::GFP (A-D), ATG‐18(FKKG)::GFP (E, F), or ATG‐18(FTTG)::GFP (G, H).


J Number of wild ‐ type or mutated ATG ‐ 18 : : GFP puncta in each strain . At least 15 embryos were scored per strain ( mean ± SD ) . * * P 0 . 0001 ; N . S : not statistically different .

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J Number of wild‐type or mutated ATG‐18::GFP puncta in each strain. At least 15 embryos were scored per strain (mean ± SD). **P 0.0001; N.S: not statistically different.


I Confocal fluorescent images of 4 ‐ fold mtm ‐ 3 embryos expressing ATG ‐ 18 : : GFP ( I ′ ) and stained by DAPI ( I ) and anti ‐ LGG ‐ 1 ( I ″ ) antibody . Insets show magnified views . Arrows indicate ATG ‐ 18 puncta labeled by anti ‐ LGG ‐ 1 antibody . Scale bars : 5 μm .

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I Confocal fluorescent images of 4‐fold mtm‐3 embryos expressing ATG‐18::GFP (I′) and stained by DAPI (I) and anti‐LGG‐1 (I″) antibody. Insets show magnified views. Arrows indicate ATG‐18 puncta labeled by anti‐LGG‐1 antibody. Scale bars: 5 μm.


Western blots for detection of TCF7 , LEF1 , TCF7L1 , TCF7L2 and β - catenin ( B " ) proteins in wild - type ( WT ) and knockout ( K . O . )

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Western blots for detection of TCF7, LEF1, TCF7L1, TCF7L2 and β-catenin (B") proteins in wild-type (WT) and knockout (K.O.)


TCF - reporter TOP - flash assay performed on the indicated cell lines treated with 10 μM CHIR , for 24 hrs . The control FOP - flash reporter is not activated upon CHIR administration . The data represent the mean ± s . e . m . of averages of nine independent experiments ( N = 9 ) . dBcat , β - catenin knockout cells ; d4TCF , TCF / LEF quadruple knockout cells Transfection of individual TCF - expressing plasmids could restore the ability of d4TCF cells to respond to Wnt3 as measured in a TOP - flash

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TCF-reporter TOP-flash assay performed on the indicated cell lines treated with 10 μM CHIR, for 24 hrs. The control FOP-flash reporter is not activated upon CHIR administration. The data represent the mean ± s.e.m. of averages of nine independent experiments (N = 9). dBcat, β-catenin knockout cells; d4TCF, TCF/LEF quadruple knockout cells Transfection of individual TCF-expressing plasmids could restore the ability of d4TCF cells to respond to Wnt3 as measured in a TOP-flash assa


Quantitative RT - PCR analyses of Axin2 transcripts , performed on the different cell lines treated with 10 μM CHIR or DMSO ( control ) for 24 hrs . Error bars show that standard deviation obtained from 3 independent experiments Transfection of individual TCF - expressing plasmids could restore the ability of d4TCF cells to respond t CHI as measured by Axin2 mRNA

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Quantitative RT-PCR analyses of Axin2 transcripts, performed on the different cell lines treated with 10 μM CHIR or DMSO (control) for 24 hrs. Error bars show that standard deviation obtained from 3 independent experiments Transfection of individual TCF-expressing plasmids could restore the ability of d4TCF cells to respond t CHI as measured by Axin2 mRNA abundance


a Heat map showing the upregulated genes in wild - type ( WT ) parental HEK 293T cells upon CHIR treatment , ranked based on the log2 fold change . Increased and decreased transcription is depicted in red and blue , respectively . Gene names are indicated along the y - axis . Canonical Wnt targets are among the most upregulated genes in WT cells ( red boxes ) . The genotypes of the cell clones used is indicated on the bottom of the heat map . Triplicates of each cell line are included . b Hierarchical clustering of differentially expressed genes with at least a 2 - fold change in any comparison . Dendrogram clustering indicates the relationships between the different experimental conditions . c Volcano plot displays differentially regulated genes in d4TCF ( C ) and dBcat ( C " ) compared to WT parental cells . Red dots indicate significantly regulated genes based on adjusted p - value and log - fold - change ( logFC ) . d Smear plots for every cell line indicating the behaviour of every gene after CHIR treatment . Differential gene expression is indicated by the log2 fold change ( logFC ) , and the average logCPM ( logarithm of counts per million reads ) was used to estimate the accuracy of the

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a Heat map showing the upregulated genes in wild-type (WT) parental HEK 293T cells upon CHIR treatment, ranked based on the log2 fold change. Increased and decreased transcription is depicted in red and blue, respectively. Gene names are indicated along the y-axis. Canonical Wnt targets are among the most upregulated genes in WT cells (red boxes). The genotypes of the cell clones used is indicated on the bottom of the heat map. Triplicates of each cell line are included. b Hierarchical clustering of differentially expressed genes with at least a 2-fold change in any comparison. Dendrogram clustering indicates the relationships between the different experimental conditions. c Volcano plot displays differentially regulated genes in d4TCF (C) and dBcat (C") compared to WT parental cells. Red dots indicate significantly regulated genes based on adjusted p-value and log-fold-change (logFC). d Smear plots for every cell line indicating the behaviour of every gene after CHIR treatment. Differential gene expression is indicated by the log2 fold change (logFC), and the average logCPM (logarithm of counts per million reads) was used to estimate the accuracy of the expression