Immunohistochemistry for active caspase ‐ 3 to detect apoptotic cells revealed a significant increase in the number of apoptotic cells in both crypt ( n = 5 , P = 0 . 0001 ) and villus ( n = 5 , P = 0 . 005 ) regions in Snai1KO tissue ( arrows mark active caspase ‐ 3 ‐ positive cells ) . Scale bars : 20 μm .

O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

Immunohistochemistry for active caspase‐3 to detect apoptotic cells revealed a significant increase in the number of apoptotic cells in both crypt (n = 5, P = 0.0001) and villus (n = 5, P = 0.005) regions in Snai1KO tissue (arrows mark active caspase‐3‐positive cells). Scale bars: 20 μm.


A lineage tracing strategy was employed to determine the fate of CBCcells that lack Snai1 . Tissue from Lgr5 ‐ EGFP ‐ IRES ‐ creERT2Snai1 + / + ( control ) and Lgr5 ‐ EGFP ‐ IRES ‐ creERT2Snai1fl / fl ( Snai1KO ) that also contained the ROSA26LacZCre reporter allele was stained with X ‐ Gal 5 days after tamoxifen injection . Quantification of X ‐ Gal ‐ positive foci per cm shows Snai1 knockout tissue failed to lineage trace indicating that no progeny are produced from Snai1 knockout cells . Bars represent mean ± SD ( n = 3 , P = 0 . 0001 ) . Scale bars : 0 . 50 mm . Two ‐ tailed Student ' s t ‐ test was used to assess significance . See also Supplementary Figure S2 .

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

A lineage tracing strategy was employed to determine the fate of CBCcells that lack Snai1. Tissue from Lgr5‐EGFP‐IRES‐creERT2Snai1+/+ (control) and Lgr5‐EGFP‐IRES‐creERT2Snai1fl/fl (Snai1KO) that also contained the ROSA26LacZCre reporter allele was stained with X‐Gal 5 days after tamoxifen injection. Quantification of X‐Gal‐positive foci per cm shows Snai1 knockout tissue failed to lineage trace indicating that no progeny are produced from Snai1 knockout cells. Bars represent mean ± SD (n = 3, P = 0.0001). Scale bars: 0.50 mm. Two‐tailed Student's t‐test was used to assess significance. See also Supplementary Figure S2.


VillinCreERT2Snai1fl / florganoids treated with tamoxifen show arrested growth after 2 and 4 days of treatment compared to controls . Scale bars : 50 μm . Quantification of viable cell growth in VillinCreERT2Snai1fl / florganoids treated with tamoxifen compared to controls over 4 days . Bars represent mean ± SD . n = 3 experiments per group , * * P = 0 . 002 .

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

VillinCreERT2Snai1fl/florganoids treated with tamoxifen show arrested growth after 2 and 4 days of treatment compared to controls. Scale bars: 50 μm.Quantification of viable cell growth in VillinCreERT2Snai1fl/florganoids treated with tamoxifen compared to controls over 4 days. Bars represent mean ± SD. n = 3 experiments per group, **P = 0.002.


Assessment of apoptosis in control and VillinCreERT2 Snai1fl / fl organoids after 4 days of growth . Photographs show an overlay of bright ‐ field and fluorescent images with propidium iodide and Hoechst staining . Quantification of apoptotic organoids . Bars represent mean ± SD . n = 3 experiments per group , * * P = 0 . 00001 . Two ‐ tailed Student ' s t ‐ test was used to assess significance . Scale bars : 50 μm . See also Supplementary Figure S3 .

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

Assessment of apoptosis in control and VillinCreERT2 Snai1fl/fl organoids after 4 days of growth. Photographs show an overlay of bright‐field and fluorescent images with propidium iodide and Hoechst staining. Quantification of apoptotic organoids. Bars represent mean ± SD. n = 3 experiments per group, **P = 0.00001. Two‐tailed Student's t‐test was used to assess significance. Scale bars: 50 μm. See also Supplementary Figure S3.


The consequences of Snai1 knockout were analysed over a time course . Immunohistochemistry for Snai1 revealed significant loss of expression at day 5 following induction compared to control . The epithelium is re ‐ populated by wild ‐ type cells as shown on day 60 . Scale bars : 20 μm .

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

The consequences of Snai1 knockout were analysed over a time course. Immunohistochemistry for Snai1 revealed significant loss of expression at day 5 following induction compared to control. The epithelium is re‐populated by wild‐type cells as shown on day 60. Scale bars: 20 μm.


A PAS staining was used to detect goblet cells in the small intestine , and no differences in cell numbers were observed in test versus control mice ( n = 4 , NS ) .

O O O O O O O O O O O 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 PAS staining was used to detect goblet cells in the small intestine, and no differences in cell numbers were observed in test versus control mice (n = 4, NS).


B Immunohistochemistry for synaptophysin was used to detect enteroendocrine cells . Quantification of the number of enteroendocrine cells per crypt - villus axes revealed a significant decrease in enteroendocrine cell numbers when Snai1 levels are elevated ( n = 4 , P = 0 . 003 ) .

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

B Immunohistochemistry for synaptophysin was used to detect enteroendocrine cells. Quantification of the number of enteroendocrine cells per crypt-villus axes revealed a significant decrease in enteroendocrine cell numbers when Snai1 levels are elevated (n = 4, P = 0.003).


C Immunohistochemistry for lysozyme was used to detect Paneth cells . Quantification of Paneth cell numbers indicated a significant decrease in Paneth cells in the base of crypts in mice with elevated Snai1 levels ( n = 5 , P = 0 . 027 ) .

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

C Immunohistochemistry for lysozyme was used to detect Paneth cells. Quantification of Paneth cell numbers indicated a significant decrease in Paneth cells in the base of crypts in mice with elevated Snai1 levels (n = 5, P = 0.027).


D , E Quantification of total PCNA ‐ positive cells per crypt revealed a significant increase when Snai1 levels were elevated ( n = 4 , P = 0 . 009 ) . This was confirmed in VillinCreERT2 ROSA26Snai1 mice 5 days after induction with tamoxifen where analysis of ( E ) PCNA ‐ positive cells in the base of crypts revealed a significant increase in proliferating cells in crypts with elevated Snai1 levels compared to control tissue ( n = 4 , P = 0 . 0005 ) . Bars represent mean ± SD .

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

D, E Quantification of total PCNA‐positive cells per crypt revealed a significant increase when Snai1 levels were elevated (n = 4, P = 0.009). This was confirmed in VillinCreERT2 ROSA26Snai1 mice 5 days after induction with tamoxifen where analysis of (E) PCNA‐positive cells in the base of crypts revealed a significant increase in proliferating cells in crypts with elevated Snai1 levels compared to control tissue (n = 4, P = 0.0005). Bars represent mean ± SD.


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


F PCNAimmunostaining ( green ) and DAPIstaining ( blue ) of VillinCreERT2ROSA26Snai1 compared to control VillinCreERT2 littermates . An expansion of proliferating CBCcells ( indicated by arrows ) is observed at the base of crypts . Scale bars : 10 μm .

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

F PCNAimmunostaining (green) and DAPIstaining (blue) of VillinCreERT2ROSA26Snai1 compared to control VillinCreERT2 littermates. An expansion of proliferating CBCcells (indicated by arrows) is observed at the base of crypts. Scale bars: 10 μm.


G qRT - PCR analysis of stem cell marker expression in the small intestine from VillinCreERT2 ROSA26Snai1 compared to control VillinCreER2T littermates . Bars represent mean ± SD . n = 3 experiments per group , Olfm4 P = 0 . 013 , Cdh1 / E ‐ cadherin P = 0 . 003 , Lgr5 P = 0 . 013 .

O O O O O O O O O O O O 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 B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O

G qRT-PCR analysis of stem cell marker expression in the small intestine from VillinCreERT2 ROSA26Snai1 compared to control VillinCreER2T littermates. Bars represent mean ± SD. n = 3 experiments per group, Olfm4 P = 0.013, Cdh1/E‐cadherin P = 0.003, Lgr5 P = 0.013.


H CD44v6 immunostaining of tissue from AhCre control compared to AhCre RosaSnai1 tissue . A significant increase in CD44v6 ‐ positive cells localised at the base of crypts ( depicted by arrows ) was observed . Bars represent mean ± SD . n = 3 mice per group , * P = 0 . 00023 . Two ‐ tailed Student ' s t ‐ test was used to assess significance . Scale bars : 10 μm .

O B-MEASURED_VAR 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

H CD44v6 immunostaining of tissue from AhCre control compared to AhCre RosaSnai1 tissue. A significant increase in CD44v6‐positive cells localised at the base of crypts (depicted by arrows) was observed. Bars represent mean ± SD. n = 3 mice per group, *P = 0.00023. Two‐tailed Student's t‐test was used to assess significance. Scale bars: 10 μm.


VillinCreERT2 RosaSnai1 organoids treated with tamoxifen show slower growth after 2 and 4 days of treatment compared to controls . Scale bars : 50 μm . Quantification of viable cells in VillinCreERT RosaSnai1 organoids treated with tamoxifen compared to control growth over 4 days . Bars represent mean ± SD . n = 3 experiments per group , * * P = 0 . 002 .

VillinCreERT2 RosaSnai1 organoids treated with tamoxifen show slower growth after 2 and 4 days of treatment compared to controls. Scale bars: 50 μm.Quantification of viable cells in VillinCreERT RosaSnai1 organoids treated with tamoxifen compared to control growth over 4 days. Bars represent mean ± SD. n = 3 experiments per group, **P = 0.002.


Assessment of apoptosis in control and VillinCreERT2 RosaSnai1 organoids after 4 days of growth . Photographs show an overlay of bright ‐ field , propidium iodide and Hoechst fluorescence . Scale bars : 50 μm . Quantification of apoptotic organoids with and without Wnt3A treatment shown as mean ± SD . n = 3 experiments per group , * * P = 0 . 00003 . Two ‐ tailed Student ' s t ‐ test was used to assess significance .

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

Assessment of apoptosis in control and VillinCreERT2 RosaSnai1 organoids after 4 days of growth. Photographs show an overlay of bright‐field, propidium iodide and Hoechst fluorescence. Scale bars: 50 μm.Quantification of apoptotic organoids with and without Wnt3A treatment shown as mean ± SD. n = 3 experiments per group, **P = 0.00003. Two‐tailed Student's t‐test was used to assess significance.


A - C Droplet digital PCR analysis of Bmf , Bbc3 / Puma and Bcl2l 11 / Bim after conditional Snai1 loss in in vitro organoid culture . VillinCreERT2 Snai1 + / + ( control ) and VillinCreERT2 Snai1fl / fl SI organoids were treated for 24 h with tamoxifen . No differences in gene expression were observed ( n = 3 ) .

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

A-C Droplet digital PCR analysis of Bmf, Bbc3/Puma and Bcl2l 11/Bim after conditional Snai1 loss in in vitro organoid culture. VillinCreERT2 Snai1+/+ (control) and VillinCreERT2 Snai1fl/fl SI organoids were treated for 24 h with tamoxifen. No differences in gene expression were observed (n = 3).


D qRT - PCR analysis of Serinc3 downregulation after conditional Snai1 loss in in vitro organoid culture . VillinCreERT2 Snai1 + / + ( control ) and VillinCreERT2 Snai1fl / fl SI organoids were treated for 24 ( P = 0 . 0001 ) and 72 h ( P = 0 . 006 ) with tamoxifen . Bars represent mean ± SD . n = 3 mice per group .

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

D qRT-PCR analysis of Serinc3 downregulation after conditional Snai1 loss in in vitro organoid culture. VillinCreERT2 Snai1+/+ (control) and VillinCreERT2 Snai1fl/fl SI organoids were treated for 24 (P = 0.0001) and 72 h (P = 0.006) with tamoxifen. Bars represent mean ± SD. n = 3 mice per group.


E SW480 cells , which transiently express GFP ‐ SNAI1 , were used to detect direct binding of SNAI1 to the promoters of known target genes CDH1 , IL8 and a novel target SERINC3 revealed by microarray analysis . An unrelated DNA region was used as a negative control . Anti ‐ GFP antibody was used to immunoprecipitate GFP ‐ tagged SNAI1 , while IgG control antibody was used as a negative control . The results are given as relative enrichment compared to input material and are representative of three independent experiments .

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

E SW480 cells, which transiently express GFP‐SNAI1, were used to detect direct binding of SNAI1 to the promoters of known target genes CDH1, IL8 and a novel target SERINC3 revealed by microarray analysis. An unrelated DNA region was used as a negative control. Anti‐GFP antibody was used to immunoprecipitate GFP‐tagged SNAI1, while IgG control antibody was used as a negative control. The results are given as relative enrichment compared to input material and are representative of three independent experiments.


Down regulation of SREBP target genes in Cideb - deficient liver . Microarray heatmap of fatty acids / cholesterol synthesis genes that were down - regulated in Cideb - / - mice and Scap liver - specific knockout mice , comparing to the respective WT controls ( WT VS Cideb - / - , WT VS L - Scap - / - , n = 2 per group ) . The expression level of each gene was linearly normalized to range from - 1 to 1 before plotting .

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

Down regulation of SREBP target genes in Cideb-deficient liver. Microarray heatmap of fatty acids/cholesterol synthesis genes that were down-regulated in Cideb-/- mice and Scap liver-specific knockout mice, comparing to the respective WT controls (WT VS Cideb-/-, WT VS L-Scap-/-, n = 2 per group). The expression level of each gene was linearly normalized to range from -1 to 1 before plotting.


Decreased SREBP processing and maturation in Cideb deficient liver . Immune blotting ( IB ) of the full length , precursor SREBP - 1 / 2 ( SREBP FL ) and the cleaved , active SREBP - 1 / 2 ( SREBP N ) from WT and Cideb - / - mice liver under ad - lib ( ND ) , fasting 12h ( F ) , and refeeding ( R ) conditions . Mice were refed with high - carbohydrate low - fat diet for 12h after 12h fasting .

O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O B-MEASURED_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 O O O O O

Decreased SREBP processing and maturation in Cideb deficient liver. Immune blotting (IB) of the full length, precursor SREBP-1/2 (SREBP FL) and the cleaved, active SREBP-1/2 (SREBP N) from WT and Cideb-/- mice liver under ad-lib (ND), fasting 12h (F), and refeeding (R) conditions. Mice were refed with high-carbohydrate low-fat diet for 12h after 12h fasting.


Defective SREBP transcriptional response to refeeding in Cideb - deficient liver . Transcript levels of lipogenic and cholesterol metabolism genes determined by qPCR from WT and Cideb - / - liver under ad - lib ( ND ) , fasting 12h ( F ) , and refeeding ( R ) conditions . Mice were refed with high - carbohydrate low - fat diet for 12h after 12h fasting ( n = 3 per group ) .

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 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

Defective SREBP transcriptional response to refeeding in Cideb-deficient liver. Transcript levels of lipogenic and cholesterol metabolism genes determined by qPCR from WT and Cideb-/- liver under ad-lib (ND), fasting 12h (F), and refeeding (R) conditions. Mice were refed with high-carbohydrate low-fat diet for 12h after 12h fasting (n = 3 per group).


Re - introduction of Cideb rescues SREBP processing in Cideb - / - liver . WT and Cideb - / - mice were injected with AAV - GFP or AAV - Cideb , and sacrificed for IB with the indicated antibodies 20 days after the injection .

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

Re-introduction of Cideb rescues SREBP processing in Cideb-/- liver. WT and Cideb-/- mice were injected with AAV-GFP or AAV-Cideb, and sacrificed for IB with the indicated antibodies 20 days after the injection.


Re - introduction of Cideb rescues the expression of SREBP target genes in Cideb - / - liver . Transcript levels of lipogenic genes determined by qPCR from WT and Cideb - / - liver . WT and Cideb - / - mice were injected with AAV - GFP or AAV - Cideb , and sacrificed for qPCR 20 days after the injection ( n = 3 per group ) .

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

Re-introduction of Cideb rescues the expression of SREBP target genes in Cideb-/- liver. Transcript levels of lipogenic genes determined by qPCR from WT and Cideb-/- liver. WT and Cideb-/- mice were injected with AAV-GFP or AAV-Cideb, and sacrificed for qPCR 20 days after the injection (n = 3 per group).


Liver morphology of WT and Cideb - / - liver under chow diet and HFLF diet . H / E staining , oil red - O staining , immunohistochemistry and ultra - structure ( Electron Microscope ) were performed . Scale bar represented 50 µm in the upper three rows of images , and 2 µm in the bottom row of images . LD : lipid droplet , ER : endoplasmic reticulum , M : mitochondrial , N : nucleus .

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

Liver morphology of WT and Cideb-/- liver under chow diet and HFLF diet. H/E staining, oil red-O staining, immunohistochemistry and ultra-structure (Electron Microscope) were performed. Scale bar represented 50 µm in the upper three rows of images, and 2 µm in the bottom row of images. LD: lipid droplet, ER: endoplasmic reticulum, M: mitochondrial, N: nucleus.


Total liver TAG levels ( B ) of WT and Cideb - / - mice under chow diet and HFLF diet ( n = 8 per group ) . Data information : Data represent Mean ± SEM ; NS : not significant , * : p < 0 . 05 , * * : p < 0 . 01 , * * * : p < 0 . 001 , by 2 - tailed Student ' s t test .

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

Total liver TAG levels (B) of WT and Cideb-/- mice under chow diet and HFLF diet (n = 8 per group). Data information: Data represent Mean ± SEM; NS: not significant, *: p < 0.05, **: p < 0.01, ***: p < 0.001, by 2-tailed Student's t test.


Liver cholesterol ester levels ( C ) of WT and Cideb - / - mice under chow diet and HFLF diet ( n = 8 per group ) . Data information : Data represent Mean ± SEM ; NS : not significant , * : p < 0 . 05 , * * : p < 0 . 01 , * * * : p < 0 . 001 , by 2 - tailed Student ' s t test .

Liver cholesterol ester levels (C) of WT and Cideb-/- mice under chow diet and HFLF diet (n = 8 per group). Data information: Data represent Mean ± SEM; NS: not significant, *: p < 0.05, **: p < 0.01, ***: p < 0.001, by 2-tailed Student's t test.


D Loss of Cideb inhibits diet - induced SREBP activation . IB of hepatic SREBP processing and lipogenic enzyme levels ( Fasn , Scd1 ) of WT and Cideb - / - mice under chow diet and HFLF diet .

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

D Loss of Cideb inhibits diet-induced SREBP activation. IB of hepatic SREBP processing and lipogenic enzyme levels (Fasn, Scd1) of WT and Cideb-/- mice under chow diet and HFLF diet.


E Serum AST and ALT levels of WT and Cideb - / - mice under chow diet and HFLF diet ( n = 8 per group ) . Data information : Data represent Mean ± SEM ; NS : not significant , * : p < 0 . 05 , * * : p < 0 . 01 , * * * : p < 0 . 001 , by 2 - tailed Student ' s t test .

O O B-MEASURED_VAR O B-MEASURED_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 O O O O O O O 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 Serum AST and ALT levels of WT and Cideb-/- mice under chow diet and HFLF diet (n = 8 per group). Data information: Data represent Mean ± SEM; NS: not significant, *: p < 0.05, **: p < 0.01, ***: p < 0.001, by 2-tailed Student's t test.


F Cideb deficiency alleviates the whole body inflammation response . Serum TNFα , MCP1 and IL6 levels of WT and Cideb - / - mice under chow diet and HFLF diet ( n = 8 per group ) . Data information : Data represent Mean ± SEM ; NS : not significant , * : p < 0 . 05 , * * : p < 0 . 01 , * * * : p < 0 . 001 , by 2 - tailed Student ' s t test .

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

F Cideb deficiency alleviates the whole body inflammation response. Serum TNFα, MCP1 and IL6 levels of WT and Cideb-/- mice under chow diet and HFLF diet (n = 8 per group). Data information: Data represent Mean ± SEM; NS: not significant, *: p < 0.05, **: p < 0.01, ***: p < 0.001, by 2-tailed Student's t test.


Serum fed glucose levels ( G ) of WT and Cideb - / - mice under chow diet and HFLF diet ( n = 8 per group ) . Data information : Data represent Mean ± SEM ; NS : not significant , * : p < 0 . 05 , * * : p < 0 . 01 , * * * : p < 0 . 001 , by 2 - tailed Student ' s t test .

Serum fed glucose levels (G) of WT and Cideb-/- mice under chow diet and HFLF diet (n = 8 per group). Data information: Data represent Mean ± SEM; NS: not significant, *: p < 0.05, **: p < 0.01, ***: p < 0.001, by 2-tailed Student's t test.


Serum fed insulin levels ( H ) of WT and Cideb - / - mice under chow diet and HFLF diet ( n = 8 per group ) . Data information : Data represent Mean ± SEM ; NS : not significant , * : p < 0 . 05 , * * : p < 0 . 01 , * * * : p < 0 . 001 , by 2 - tailed Student ' s t test .

O O B-MEASURED_VAR 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

Serum fed insulin levels (H) of WT and Cideb-/- mice under chow diet and HFLF diet (n = 8 per group). Data information: Data represent Mean ± SEM; NS: not significant, *: p < 0.05, **: p < 0.01, ***: p < 0.001, by 2-tailed Student's t test.


I Glucose tolerance test ( GTT ) of WT and Cideb - / - mice under HFLF diet ( n = 5 per group ) . Data information : Data represent Mean ± SEM ; NS : not significant , * : p < 0 . 05 , * * : p < 0 . 01 , * * * : p < 0 . 001 , by 2 - tailed Student ' s t test .

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

I Glucose tolerance test (GTT) of WT and Cideb-/- mice under HFLF diet (n = 5 per group). Data information: Data represent Mean ± SEM; NS: not significant, *: p < 0.05, **: p < 0.01, ***: p < 0.001, by 2-tailed Student's t test.


J Insulin tolerance test ( ITT ) of WT and Cideb - / - mice under HFLF diet ( n = 5 per group ) . Data information : Data represent Mean ± SEM ; NS : not significant , * : p < 0 . 05 , * * : p < 0 . 01 , * * * : p < 0 . 001 , by 2 - tailed Student ' s t test .

J Insulin tolerance test (ITT) of WT and Cideb-/- mice under HFLF diet (n = 5 per group). Data information: Data represent Mean ± SEM; NS: not significant, *: p < 0.05, **: p < 0.01, ***: p < 0.001, by 2-tailed Student's t test.


Decreased SREBP - 1 / 2 and SCAP levels in Golgi apparatus in Cideb - / - mice . IB of organelle fractions of the indicated proteins and quantification of IB data from 3 independent experiments . Data information : Data represent the Mean ± SEM ; NS : not significant , * : p < 0 . 05 , * * : p < 0 . 01 , * * * : p < 0 . 001 , by 2 - tailed Student ' s t test .

O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR 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

Decreased SREBP-1/2 and SCAP levels in Golgi apparatus in Cideb-/- mice. IB of organelle fractions of the indicated proteins and quantification of IB data from 3 independent experiments. Data information: Data represent the Mean ± SEM; NS: not significant, *: p < 0.05, **: p < 0.01, ***: p < 0.001, by 2-tailed Student's t test.


Cideb deficiency impairs the packaging of SCAP / SREBP into COPII vesicles . Protein present in the vesicle or microsome fractions obtained from WT or Cideb - / - liver were detected by IB . Quantification of IB data from 3 independent experiments . Data information : Data represent the Mean ± SEM ; NS : not significant , * : p < 0 . 05 , * * : p < 0 . 01 , * * * : p < 0 . 001 , by 2 - tailed Student ' s t test .

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

Cideb deficiency impairs the packaging of SCAP/SREBP into COPII vesicles. Protein present in the vesicle or microsome fractions obtained from WT or Cideb-/- liver were detected by IB. Quantification of IB data from 3 independent experiments. Data information: Data represent the Mean ± SEM; NS: not significant, *: p < 0.05, **: p < 0.01, ***: p < 0.001, by 2-tailed Student's t test.


Cideb depletion decreases the recruitment of SCAP to the COPII machinery in cells . Primary hepatocytes transfected with control or Cideb siRNA were infected with adenovirus expressing Sar1A - Flag - BirA * . Cells were cultured in sterol - depleted medium and treated with 80 µM biotin , prior to pulldown with streptavidin - conjugated beads . The biotinylated proteins were detected by IB with the indicated antibodies following SDS - PAGE .

B-CONTROLLED_VAR O O O O O B-MEASURED_VAR 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 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

Cideb depletion decreases the recruitment of SCAP to the COPII machinery in cells. Primary hepatocytes transfected with control or Cideb siRNA were infected with adenovirus expressing Sar1A-Flag-BirA*. Cells were cultured in sterol-depleted medium and treated with 80 µM biotin, prior to pulldown with streptavidin-conjugated beads. The biotinylated proteins were detected by IB with the indicated antibodies following SDS-PAGE.


Cleaved SREBP bypasses Cideb deficiency . WT and Cideb - / - mice were injected with adenovirus expressing GFP , SREBP - 1c full length ( FL ) or SREBP - 1c active form ( N ) . Mice were sacrificed 7 days after injection , and liver TAG levels and hepatic expression SREBP - 1 target genes in the indicated groups were determined ( n = 5 per group ) . Data information : Data represent the Mean ± SEM ; NS : not significant , * : p < 0 . 05 , * * : p < 0 . 01 , * * * : p < 0 . 001 , by 2 - tailed Student ' s t test .

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

Cleaved SREBP bypasses Cideb deficiency. WT and Cideb-/- mice were injected with adenovirus expressing GFP, SREBP-1c full length (FL) or SREBP-1c active form (N). Mice were sacrificed 7 days after injection, and liver TAG levels and hepatic expression SREBP-1 target genes in the indicated groups were determined (n=5 per group). Data information: Data represent the Mean ± SEM; NS: not significant, *: p < 0.05, **: p < 0.01, ***: p < 0.001, by 2-tailed Student's t test.


Cideb co - precipitates with SCAP . Flag - tagged Cideb was co - expressed with different HA - tagged proteins ( PLIN2 , Insig , SCAP , SREBP - 1 or SREBP - 2 ) in 293T cells and immuno - precipitated with an anti - Flag antibody , and the co - immunoprecipitated protein were detected by an anti - HA antibody following SDS - PAGE .

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

Cideb co-precipitates with SCAP. Flag-tagged Cideb was co-expressed with different HA-tagged proteins (PLIN2, Insig, SCAP, SREBP-1 or SREBP-2) in 293T cells and immuno-precipitated with an anti-Flag antibody, and the co-immunoprecipitated protein were detected by an anti-HA antibody following SDS-PAGE.


Interaction between the endogenous SCAP and Cideb . Endogenous SCAP was immuno - precipitated from primary hepatocytes under normal medium , 25 - HC depletion or addition conditions . And the presence of Cideb in the immune - complex was detected by IB . SREBP - 1 was detected as the positive control . Grp94 was detected as the negative control .

O O O O B-MEASURED_VAR O B-MEASURED_VAR 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 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

Interaction between the endogenous SCAP and Cideb. Endogenous SCAP was immuno-precipitated from primary hepatocytes under normal medium, 25-HC depletion or addition conditions. And the presence of Cideb in the immune-complex was detected by IB. SREBP-1 was detected as the positive control. Grp94 was detected as the negative control.


Cideb interacts with the 416 - 800 portion of SCAP . Flag - tagged Cideb was co - expressed with the indicated HA - tagged SCAP truncation mutants in 293T cells and immuno - precipitated with an anti - Flag antibody . Levels of the co - immunoprecipitated protein were detected by an anti - HA antibody following SDS - PAGE .

B-MEASURED_VAR 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 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

Cideb interacts with the 416-800 portion of SCAP. Flag-tagged Cideb was co-expressed with the indicated HA-tagged SCAP truncation mutants in 293T cells and immuno-precipitated with an anti-Flag antibody. Levels of the co-immunoprecipitated protein were detected by an anti-HA antibody following SDS-PAGE.


Sterol - dependent interaction switch of SCAP . SCAP interacts with Insig in the presence of sterol , while switches to Cideb binding upon sterol deprivation . Flag - tagged Insig - 1 or Cideb was co - expressed with HA - tagged SREBP - 2 and SCAP or SCAP - Y298C in CHO - K1 cells in sterol - depleted medium . When indicated , 10 µg / ml 25 - HC were supplemented before cells were immuno - precipitated with an anti - Flag antibody , and the levels of co - immunoprecipitated proteins were detected by IB following SDS - PAGE . The dotted blue vertical line was drawn to separate the result between SCAP - WT and SCAP - Y298C .

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

Sterol-dependent interaction switch of SCAP. SCAP interacts with Insig in the presence of sterol, while switches to Cideb binding upon sterol deprivation. Flag-tagged Insig-1 or Cideb was co-expressed with HA-tagged SREBP-2 and SCAP or SCAP-Y298C in CHO-K1 cells in sterol-depleted medium. When indicated, 10 µg/ml 25-HC were supplemented before cells were immuno-precipitated with an anti-Flag antibody, and the levels of co-immunoprecipitated proteins were detected by IB following SDS-PAGE. The dotted blue vertical line was drawn to separate the result between SCAP-WT and SCAP-Y298C.


Cideb relieves sterol - dependent suppression of SREBP processing . Hepatocytes infected with adenovirus expressing GFP or Flag - Cideb were cultured in sterol deprived condition . When indicated , cells were treated with different doses of 25 - HC before subjected to IB with the indicated antibodies following SDS - PAGE . Quantification of IB data from 3 independent experiments . Data represents Mean ± SEM .

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

Cideb relieves sterol-dependent suppression of SREBP processing. Hepatocytes infected with adenovirus expressing GFP or Flag-Cideb were cultured in sterol deprived condition. When indicated, cells were treated with different doses of 25-HC before subjected to IB with the indicated antibodies following SDS-PAGE. Quantification of IB data from 3 independent experiments. Data represents Mean ± SEM.


Cideb promotes Golgi localization of SCAP . HepG2 cells transfected with GFP - SCAP alone or with HA - Cideb were cultured in sterol deprived conditions or treated with 1 µg / ml 25 - HC . Cells were fixed and subjected to confocal microscopy following staining with antibodies against GM130 or HA . Scale bars represent 10 µm ( Merge ) , and 2 µm ( Inlay ) . Bottom : quantification of the percentage of SCAP localized in Golgi - positive region per cell ( left ) and quantification of the percentage of cells containing Golgi - localized GFP - SCAP ( right ) under indicated conditions , from 3 independent experiments ; Data represent Mean ± SEM ; * * : p < 0 . 01 , * * * : p < 0 . 001 , by 2 - tailed Student ' s t test .

B-CONTROLLED_VAR O O O O B-MEASURED_VAR 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 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 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 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

Cideb promotes Golgi localization of SCAP. HepG2 cells transfected with GFP-SCAP alone or with HA-Cideb were cultured in sterol deprived conditions or treated with 1 µg/ml 25-HC. Cells were fixed and subjected to confocal microscopy following staining with antibodies against GM130 or HA. Scale bars represent 10 µm (Merge), and 2 µm (Inlay). Bottom: quantification of the percentage of SCAP localized in Golgi-positive region per cell (left) and quantification of the percentage of cells containing Golgi-localized GFP-SCAP (right) under indicated conditions, from 3 independent experiments; Data represent Mean ± SEM; **: p < 0.01, ***: p < 0.001, by 2-tailed Student's t test.


Cideb interacts with Sec12 . 293T cells transfected with Cideb and GFP - tagged Sec12 , Sec23 , Sec16S , or Sec16L were subjected to immuno - precipitation with an anti - Cideb antibody , and levels of the co - immunoprecipitated protein were detected by an anti - GFP antibody .

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

Cideb interacts with Sec12. 293T cells transfected with Cideb and GFP-tagged Sec12, Sec23, Sec16S, or Sec16L were subjected to immuno-precipitation with an anti-Cideb antibody, and levels of the co-immunoprecipitated protein were detected by an anti-GFP antibody.


Co - localization of SCAP and Cideb at the ER exit sites . HepG2 cells transfected with GFP - SCAP and HA - Cideb were arrested with 10 µM BFA and 1 µM Nocodazole for 2 hours , then fixed with methanol and stained with antibodies against Sec12 or HA . 3D reconstruction was obtained by processing 3D - original image with Imaris . Scale bars represent 10 µm ( Merge ) , and 1 µm ( Inlay and 3D reconstruction ) .

O O O O B-MEASURED_VAR O B-MEASURED_VAR 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 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

Co-localization of SCAP and Cideb at the ER exit sites. HepG2 cells transfected with GFP-SCAP and HA-Cideb were arrested with 10 µM BFA and 1 µM Nocodazole for 2 hours, then fixed with methanol and stained with antibodies against Sec12 or HA. 3D reconstruction was obtained by processing 3D-original image with Imaris. Scale bars represent 10 µm (Merge), and 1 µm (Inlay and 3D reconstruction).


Localization of Cideb at the ER exit sites . AML12 cells knocked - in with a GFP tag at the C - terminus of Cideb genome was treated with 10 µM BFA for 1 hour , then fixed with methanol and stained with antibodies against Sec12 . Scale bars represent 10 µm ( Merge ) , and 1 µm ( Inlay ) .

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 B-CONTROLLED_VAR 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

Localization of Cideb at the ER exit sites. AML12 cells knocked-in with a GFP tag at the C-terminus of Cideb genome was treated with 10 µM BFA for 1 hour, then fixed with methanol and stained with antibodies against Sec12. Scale bars represent 10 µm (Merge), and 1 µm (Inlay).


Sec12 interacts with the 118 - 165 portion of Cideb . HA - tagged Sec12 was co - expressed with the indicated Flag - tagged Cideb truncation mutants in 293T cells and immuno - precipitated with an anti - Flag antibody . Levels of the co - immunoprecipitated protein were detected by an anti - HA antibody following SDS - PAGE .

Sec12 interacts with the 118-165 portion of Cideb. HA-tagged Sec12 was co-expressed with the indicated Flag-tagged Cideb truncation mutants in 293T cells and immuno-precipitated with an anti-Flag antibody. Levels of the co-immunoprecipitated protein were detected by an anti-HA antibody following SDS-PAGE.


Lysine 128 of Cideb is required for the interaction with Sec12 . 293T cells transfected with HA - Sec12 and the indicated Flag - tagged Cideb constructs were subjected to immunoprecipitation with an anti - Flag antibody , and levels of the co - immunoprecipitated HA - Sec12 were detected with an anti - HA antibody .

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

Lysine 128 of Cideb is required for the interaction with Sec12. 293T cells transfected with HA-Sec12 and the indicated Flag-tagged Cideb constructs were subjected to immunoprecipitation with an anti-Flag antibody, and levels of the co-immunoprecipitated HA-Sec12 were detected with an anti-HA antibody.


Recombinant Cideb interacts with Sec12 . GST - Sec12 and MBP - tagged Cideb or Cideb - K128A were purified from E . coli and subjected to in vitro binding .

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

Recombinant Cideb interacts with Sec12. GST-Sec12 and MBP-tagged Cideb or Cideb-K128A were purified from E.coli and subjected to in vitro binding.


Cideb - K128A fails to promote SREBP processing . Cideb - / - mice were injected with adenovirus expressing GFP , Flag - Cideb , or Flag - Cideb - K128A , and sacrificed 7 days after injection . Liver lysates were subjected to IB with the indicated antibodies following SDS - PAGE and quantification of IB data from 3 independent experiments . Data represent Mean ± SEM ; * * : p < 0 . 01 , * * * : p < 0 . 001 , by 2 - tailed Student ' s t test .

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

Cideb-K128A fails to promote SREBP processing. Cideb-/- mice were injected with adenovirus expressing GFP, Flag-Cideb, or Flag-Cideb-K128A, and sacrificed 7 days after injection. Liver lysates were subjected to IB with the indicated antibodies following SDS-PAGE and quantification of IB data from 3 independent experiments. Data represent Mean ± SEM; **: p < 0.01, ***: p < 0.001, by 2-tailed Student's t test.


Cideb , but not Cideb - K128A , promotes SCAP / SREBP incorporation into COPII vesicles . Liver microsomes were isolated from mice expressing the indicated proteins and subjected to in vitro budding assay . Resulting vesicles and the donor microsomes were subject to IB with the indicated antibodies following SDS - PAGE .

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

Cideb, but not Cideb-K128A, promotes SCAP/SREBP incorporation into COPII vesicles. Liver microsomes were isolated from mice expressing the indicated proteins and subjected to in vitro budding assay. Resulting vesicles and the donor microsomes were subject to IB with the indicated antibodies following SDS-PAGE.


Cideb enriches SCAP to ERESs via interacting with Sec12 . CHO - K1 cells transfected with GFP - SCAP , HA - Cideb or HA - Cideb - K128A were arrested with 5 µM Nocodazole for 30 min , then fixed with methanol and stained with antibodies against Sec12 and HA . Scale bars represent 10 µm ( left ) , and 2 . 5 µm ( Inlay ) .

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

Cideb enriches SCAP to ERESs via interacting with Sec12. CHO-K1 cells transfected with GFP-SCAP, HA-Cideb or HA-Cideb-K128A were arrested with 5 µM Nocodazole for 30 min, then fixed with methanol and stained with antibodies against Sec12 and HA. Scale bars represent 10 µm (left), and 2.5 µm (Inlay).


Number of SCAP - positive ER exit sites per cell . Normalized fluorescent intensity of SCAP at each individual ER exit site . Total fluorescent intensity of SCAP at ER exit sites per cell . Number of Sec12 - positive ER exit sites per cell . Data information : Data represent Mean ± SEM ; NS : not significant , * : p < 0 . 05 , * * : p < 0 . 01 , * * * : p < 0 . 001 , by 2 - tailed Student ' s t test . In panel C - F , quantification of images from 3 independent experiments .

O O B-MEASURED_VAR 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 B-MEASURED_VAR 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

Number of SCAP-positive ER exit sites per cell.   Normalized fluorescent intensity of SCAP at each individual ER exit site.   Total fluorescent intensity of SCAP at ER exit sites per cell.   Number of Sec12-positive ER exit sites per   cell. Data information: Data represent Mean ± SEM; NS: not significant, *: p < 0.05, **: p < 0.01, ***: p < 0.001, by 2-tailed Student's t test. In panel C-F, quantification of images from 3 independent experiments.


HeLa cells were transfected with a WT or mutant Parkin plasmid ( FLAG ) , shown in blue , along with reporters for either PA or DAG shown in green . DMSO or 10 µM CCCP was added for the indicated periods of time . Mitochondria were visualized via anti - Tom20 staining ( red ) . ( A ) PA accumulated on Parkin - positive mitochondria after 1 hour of 10uM CCCP treatment , with a more dramatic increase by 3hr and 18 hr . Scale bar = 25 µM , and zoom is 9x . ( B ) Quantification of imaging experiments shown in A and B . Bars represent mean with SEM from three biological independent experiments ; two - way ANOVA analysis was performed for statistical analysis ( * * * P < 0 . 0001 ) . ( C ) DAG ( diacylglycerol ) accumulated on Parkin - positive mitochondria after 5 . 5 hr of 10uM CCCP treatment . Arrowheads and arrows point to the DAGR concentration at the Golgi and mitochondria , respectively . Scale bar = 25 µM , and zoom is 9x .

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 B-MEASURED_VAR 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 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

HeLa cells were transfected with a WT or mutant Parkin plasmid (FLAG), shown in blue, along with reporters for either PA or DAG shown in green. DMSO or 10 µM CCCP was added for the indicated periods of time. Mitochondria were visualized via anti-Tom20 staining (red). (A) PA accumulated on Parkin-positive mitochondria after 1 hour of 10uM CCCP treatment, with a more dramatic increase by 3hr and 18 hr. Scale bar = 25 µM, and zoom is 9x. (B) Quantification of imaging experiments shown in A and B. Bars represent mean with SEM from three biological independent experiments; two-way ANOVA analysis was performed for statistical analysis (***P<0.0001). (C) DAG (diacylglycerol) accumulated on Parkin-positive mitochondria after 5.5 hr of 10uM CCCP treatment. Arrowheads and arrows point to the DAGR concentration at the Golgi and mitochondria, respectively. Scale bar = 25 µM, and zoom is 9x.


( B ) HEK - 293T cells were transfected with Parkin - FLAG and DAGR - YFP followed by CCCP treatment for indicated times . Mitochondrial and cytosolic fractions were purified and analyzed by Western blots by indicated antibodies . Citrate synthetase and α - tubulin were used as a mitochondrial and cytosolic marker , respectively . Note that PLD2 and Parkin levels were elevated in the mitochondrial fraction in response to CCCP treatment .

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

(B) HEK-293T cells were transfected with Parkin-FLAG and DAGR-YFP followed by CCCP treatment for indicated times. Mitochondrial and cytosolic fractions were purified and analyzed by Western blots by indicated antibodies. Citrate synthetase and α-tubulin were used as a mitochondrial and cytosolic marker, respectively. Note that PLD2 and Parkin levels were elevated in the mitochondrial fraction in response to CCCP treatment.


( C ) Hela cells were co - transfected with mCherry - Parkin and the PA reporter , followed by CCCP treatment alone or with a PLD2 inhibitor VU 0364739 ( 3 µM ) for 5 . 5 hrs . Line scan analysis ( Image J software ) corresponding to the line drawn in the images indicate colocalization between the PA reporter ( green ) and mitochondria ( red ) . Note that VU 0364739 suppressed mitochondrial PA accumulation . Scale bar = 10 µM . ( D ) Quantification of the numbers of cells with the PA reporter positive mitochondria shown in ( C ) . Asterisks indicate statistical significance ( * * * P < 0 . 001 , Student ' s t - test ) from three biological independent experiments . The bars indicate mean ± SEM .

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

(C) Hela cells were co-transfected with mCherry-Parkin and the PA reporter, followed by CCCP treatment alone or with a PLD2 inhibitor VU 0364739 (3 µM) for 5.5 hrs. Line scan analysis (Image J software) corresponding to the line drawn in the images indicate colocalization between the PA reporter (green) and mitochondria (red). Note that VU 0364739 suppressed mitochondrial PA accumulation. Scale bar = 10 µM. (D) Quantification of the numbers of cells with the PA reporter positive mitochondria shown in (C). Asterisks indicate statistical significance (***P<0.001, Student's t-test) from three biological independent experiments. The bars indicate mean± SEM.


( E ) Hela cells were transfected with a lipin1 siRNA , followed by the DAG reporter and mCherry - Parkin , treated with CCCP ( 10 µM ) for 9 hrs and then subject to image analysis . Scale bar = 25 um , and zoom is 4x . Lipin 1 KD reduced mitochondrial DAGR , but not Golgi - DAGR , accumulation ( arrowheads ) ( F ) The numbers of cells with DAG - reporter positive mitochondria shown in ( E ) and in Lipin - 1 knockdown cells transfected with siRNA - resistant wildtype ( WT ) and catalytically dead ( CD ; D712E ; D714E ) mutant cDNA . Note that Lipin1 KD reduced mitochondrial DAG accumulation , which can be significantly restored by the re - expression of WT , but not CD mutant , Lipin - 1 . The graph shows the means with SEM ( error bars ) from three biological independent experiments . Asterisks indicate statistical significance by one - way ANOVA ( * * P < 0 . 01 , * * * P < 0 . 001 ) .

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

(E) Hela cells were transfected with a lipin1 siRNA, followed by the DAG reporter and mCherry-Parkin, treated with CCCP (10 µM) for 9 hrs and then subject to image analysis. Scale bar = 25 um, and zoom is 4x. Lipin 1 KD reduced mitochondrial DAGR, but not Golgi-DAGR, accumulation (arrowheads) (F) The numbers of cells with DAG-reporter positive mitochondria shown in (E) and in Lipin-1 knockdown cells transfected with siRNA-resistant wildtype (WT) and catalytically dead (CD; D712E;D714E) mutant cDNA. Note that Lipin1 KD reduced mitochondrial DAG accumulation, which can be significantly restored by the re-expression of WT, but not CD mutant, Lipin-1. The graph shows the means with SEM (error bars) from three biological independent experiments. Asterisks indicate statistical significance by one-way ANOVA (**P< 0.01, ***P<0.001).


( A - B ) Hela cells were transfected with a Lipin1 or control ( cKD ) siRNA followed by a GFP - Parkin expression plasmid . Transfected cells were treated with CCCP at 10 µM for 18 hrs , and subject to immuno - staining . ( B ) Quantification of Parkin - positive cells in ( A ) that have lost a majority of mitochondria ( marked by TOM20 , arrows ) . Note that mitochondrial clearance is reduced in Lipin - 1 knockdown cells ( arrowheads in ( A ) ) . Scale bar = 25 µM .

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

(A-B) Hela cells were transfected with a Lipin1 or control (cKD) siRNA followed by a GFP-Parkin expression plasmid. Transfected cells were treated with CCCP at 10 µM for 18 hrs, and subject to immuno-staining. (B) Quantification of Parkin-positive cells in (A) that have lost a majority of mitochondria (marked by TOM20, arrows). Note that mitochondrial clearance is reduced in Lipin-1 knockdown cells (arrowheads in (A)). Scale bar = 25 µM.


( C ) . Control or lipin1 knockdown Hela cells stably expressing parkin - mCherry were treated with DMSO or CCCP ( 10 µM for 18 hrs ) and Bafilomycin A1 ( 1μM , lysosomal inhibitor ) treatment , followed by immunoblotting with indicated antibodies and quantified by the Image J . software . Note that lipin1 silencing suppressed CCCP - induced mitochondrial protein degradation . Lipin - 1 - mediated mitochondrial DAG production is required for mitophagosome production .

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

(C). Control or lipin1 knockdown Hela cells stably expressing parkin-mCherry were treated with DMSO or CCCP (10 µM for 18 hrs) and Bafilomycin A1 (1μM, lysosomal inhibitor) treatment, followed by immunoblotting with indicated antibodies and quantified by the Image J. software. Note that lipin1 silencing suppressed CCCP-induced mitochondrial protein degradation. Lipin-1-mediated mitochondrial DAG production is required for mitophagosome production.


( D - F ) . Hela cells were transfected with control or a Lipin1 siRNA followed by the expression plasmids of GFP - Parkin and RFP - DAGR . Transfected cells were treated with DMSO or CCCP at 10 µM for 9 hr , with or without further incubation with 1 , 2 - Dipalmitoyl - sn - glycerol ( DPG ) at 100 µM , as indicated . Autophagosome formation was assessed by an LC3 antibody ( pseudocolored in white in single - channel and blue in the overlay images ) . Arrows point to DAGR concentration at the Golgi under basal conditions . The number ( E ) and size ( F , arbitrary unit ) of LC3 vesicles were quantified . n = 4 biological replicates . The bars indicates mean ± SEM . Asterisks indicate statistical significance by one - way ANOVA ( * P < 0 . 05 , * * P < 0 . 01 ) . Scale bar = 25 µM and zoom is 5x . Note that both the number and size of LC3 - vesicles in Lipin - 1 knockdown cells were much smaller than those in WT cells , and these defects were corrected by DPG treatment .

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

(D-F). Hela cells were transfected with control or a Lipin1 siRNA followed by the expression plasmids of GFP-Parkin and RFP-DAGR. Transfected cells were treated with DMSO or CCCP at 10 µM for 9 hr, with or without further incubation with 1,2-Dipalmitoyl-sn-glycerol (DPG) at 100 µM, as indicated. Autophagosome formation was assessed by an LC3 antibody (pseudocolored in white in single-channel and blue in the overlay images). Arrows point to DAGR concentration at the Golgi under basal conditions. The number (E) and size (F, arbitrary unit) of LC3 vesicles were quantified. n=4 biological replicates. The bars indicates mean± SEM. Asterisks indicate statistical significance by one-way ANOVA (*P< 0.05, **P<0.01). Scale bar = 25 µM and zoom is 5x. Note that both the number and size of LC3-vesicles in Lipin-1 knockdown cells were much smaller than those in WT cells, and these defects were corrected by DPG treatment.


( A ) E3 ligase - deficient Parkin mutants ( T240R and T415N ) have no effect on PA accumulation ( green ) on depolarized , Parkin - positive mitochondria . The expression of WT and Parkin mutants were determined by Immunoblotting . Scale bar = 25 µM , and zoom is 3x . ( B ) DAG accumulation depends upon intact Parkin E3 ligase activity . Scale bar = 25µm , and zoom is 3x . ( C ) Quantification of imaging experiments shown in ( A ) and ( B ) . Bars represent mean with SEM ; two - way ANOVA analysis was performed for statistical analysis ( * * * P < 0 . 001 ) . n = 3 biological replicates .

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

(A) E3 ligase-deficient Parkin mutants (T240R and T415N) have no effect on PA accumulation (green) on depolarized, Parkin-positive mitochondria. The expression of WT and Parkin mutants were determined by Immunoblotting. Scale bar = 25 µM, and zoom is 3x. (B) DAG accumulation depends upon intact Parkin E3 ligase activity. Scale bar = 25µm, and zoom is 3x. (C) Quantification of imaging experiments shown in (A) and (B). Bars represent mean with SEM; two-way ANOVA analysis was performed for statistical analysis(***P<0.001). n=3 biological replicates.


( D ) Hela cells were transfected with control or optineurin - and NDP52 - siRNAs and expression plasmids of FLAG - Parkin and YFP - DAGR . These cells were treated with DMSO or 10 µM CCCP for 5 . 5 hrs , as indicated . Note that OPTN / NDP52 double knockdown prevented mitochondrial YFP - DAGR accumulation . ( Scale bar = 25 µM and zoom is 3x ) ( E ) Hela OPTN / NDP52 knockdown cells were transfected with a siRNA - resistant wildtype or ubiquitin - binding deficient E478G OPTN mutant , as indicated . The percentage of cells with mitochondrial YFP - DAGR following CCCP treatment was quantified . Asterisks indicate statistical significance by one - way ANOVA ( * * P < 0 . 01 , * * * P < 0 . 001 ) from three biological independent experiments . The bars indicate mean ± SEM . Bottom panel : Expression of OPTN , OPTN E478 and NDP52 was determined by immunoblotting .

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

(D) Hela cells were transfected with control or optineurin- and NDP52- siRNAs and expression plasmids of FLAG-Parkin and YFP-DAGR. These cells were treated with DMSO or 10 µM CCCP for 5.5 hrs, as indicated. Note that OPTN/NDP52 double knockdown prevented mitochondrial YFP-DAGR accumulation. (Scale bar = 25 µM and zoom is 3x) (E) Hela OPTN/NDP52 knockdown cells were transfected with a siRNA-resistant wildtype or ubiquitin-binding deficient E478G OPTN mutant, as indicated. The percentage of cells with mitochondrial YFP-DAGR following CCCP treatment was quantified. Asterisks indicate statistical significance by one-way ANOVA (**P< 0.01, ***P<0.001) from three biological independent experiments. The bars indicate mean± SEM. Bottom panel: Expression of OPTN, OPTN E478 and NDP52 was determined by immunoblotting.


( F - G ) Hela OPTN / NDP52 knockdown cells were incubated with 1 , 2 - Dipalmitoyl - sn - glycerol ( DPG ) followed by CCCP treatment for 18 hrs . Mitophagy efficiency was assessed by TOM20 staining and quantified in ( G ) ( n = 2 biological replicates ) . Note that DPG significantly restored mitophagy in OPTN / NDP52 knockdown cells . Scale bar = 10 µM .

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

(F-G) Hela OPTN/NDP52 knockdown cells were incubated with 1,2-Dipalmitoyl-sn-glycerol (DPG) followed by CCCP treatment for 18 hrs. Mitophagy efficiency was assessed by TOM20 staining and quantified in (G) (n = 2 biological replicates). Note that DPG significantly restored mitophagy in OPTN/NDP52 knockdown cells. Scale bar = 10 µM.


( B ) Mitochondrial and cytosolic fractions obtained from control and CCCP treated cells , were immunoblotted for EndoB1 and OPTN , as indicated .

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

(B) Mitochondrial and cytosolic fractions obtained from control and CCCP treated cells, were immunoblotted for EndoB1 and OPTN, as indicated.


( C ) . Cytosolic and mitochondrial fractions purified from control and OPTN / NDP52 knockdown cells treated with CCCP were immunoblotted with indicated antibodies . Note that mitochondrial EndoB1 levels were reduced in OPTN / NDP52 knockdown

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

(C). Cytosolic and mitochondrial fractions purified from control and OPTN/NDP52 knockdown cells treated with CCCP were immunoblotted with indicated antibodies. Note that mitochondrial EndoB1 levels were reduced in OPTN/NDP52 knockdown cells


( D - E ) Hela cells were transfected with an EndoB1 siRNA , followed by the expression plasmids for mCherry - Parkin and YFP - DAGR , and CCCP treatment ( 10 µM ) for 9 hrs . Cells with mitochondrial YFP - DAGR were quantified in ( E ) ( * P < 0 . 05 , Student ' s t - test , n = 3 biological replicates ) . Scale bar = 25 µM and zoom is 5x . Note that knockdown of EndoB1 suppressed mitochondrial DAG production .

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 B-CONTROLLED_VAR O O O O O

(D-E) Hela cells were transfected with an EndoB1 siRNA, followed by the expression plasmids for mCherry-Parkin and YFP-DAGR, and CCCP treatment (10 µM) for 9 hrs. Cells with mitochondrial YFP-DAGR were quantified in (E) (*P<0.05, Student's t-test, n=3 biological replicates). Scale bar = 25 µM and zoom is 5x. Note that knockdown of EndoB1 suppressed mitochondrial DAG production.


( B ) Detection of WASH gene targeting . The floxed WASH gene by gene targeting was analysed by Southern blotting ( left panel ) and PCR ( middle panel ) . The genotypes of the offspring were analysed by PCR ( right panel ) .

O O O O O B-MEASURED_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

(B) Detection of WASH gene targeting. The floxed WASH gene by gene targeting was analysed by Southern blotting (left panel) and PCR (middle panel). The genotypes of the offspring were analysed by PCR (right panel).


( C ) WASH deficiency is embryonic lethal . E11 . 5 of WASH deficiency showed a shrinked decidua .

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

(C) WASH deficiency is embryonic lethal. E11.5 of WASH deficiency showed a shrinked decidua.


( D ) Histology analysis of E7 . 5 WASH + / + and WASH − / − embryos . Scale bar , 100 μm .

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

(D) Histology analysis of E7.5 WASH+/+ and WASH−/−embryos. Scale bar, 100 μm.


( E ) WASH ‐ deficient embryos do not undergo apoptosis . TUNEL assay was performed for apoptosis detection . PI , propidium iodide . FITC , fluoresceinisothiocyanate . Scale bar , 100 μm .

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

(E) WASH‐deficient embryos do not undergo apoptosis. TUNEL assay was performed for apoptosis detection. PI, propidium iodide. FITC, fluoresceinisothiocyanate. Scale bar, 100 μm.


( F ) E7 . 5embryos of WASH − / − mice present extensive autophagy . Ultrastructure of isolated E7 . 5embryos was stained with antibody against LC3 and visualized by immuno ‐ electron microscopy . Black arrowhead indicates autophagosome . * , Cellular reference . Scale bar , 500 nm ( upper panel ) . WASH + / + or WASH − / − embryos were isolated , and LC3 conversion and p62 level were analysed by immunoblotting ( lower panel ) . Bands were quantified and analysed by Image J and shown as means ± s . d . * * * P0 . 001 . Above experiments were repeated for three independent times with similar results . Source data for this figure is available on the online supplementary information page .

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

(F) E7.5embryos of WASH−/−mice present extensive autophagy. Ultrastructure of isolated E7.5embryos was stained with antibody against LC3 and visualized by immuno‐electron microscopy. Black arrowhead indicates autophagosome. *, Cellular reference. Scale bar, 500 nm (upper panel). WASH+/+ or WASH−/−embryos were isolated, and LC3 conversion and p62 level were analysed by immunoblotting (lower panel). Bands were quantified and analysed by Image J and shown as means±s.d. ***P0.001. Above experiments were repeated for three independent times with similar results.Source data for this figure is available on the online supplementary information page.


( A ) WASH deficiency enhances autophagy induction . WASH + / + or WASH − / − MEFs were treated with EBSS for the indicated times in the presence or absence of 20 nM bafilomycin A1 ( BafA1 ) , and harvested for immunoblotting . Ratios of LC3 ‐ II / β ‐ actin were calculated and shown at the right panel .

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

(A) WASH deficiency enhances autophagy induction. WASH+/+ or WASH−/− MEFs were treated with EBSS for the indicated times in the presence or absence of 20 nM bafilomycin A1 (BafA1), and harvested for immunoblotting. Ratios of LC3‐II/β‐actin were calculated and shown at the right panel.


( B ) WASH knockout accelerates LC3 conversion by confocal microscopy . Endogenous LC3 puncta were visualized by staining with antibody against LC3 ( left panel ) , and calculated as shown in the right panel . Nuclei were stained with DAPI . Scale bar , 10 μm .

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

(B) WASH knockout accelerates LC3 conversion by confocal microscopy. Endogenous LC3 puncta were visualized by staining with antibody against LC3 (left panel), and calculated as shown in the right panel. Nuclei were stained with DAPI. Scale bar, 10 μm.


( C ) Poly ‐ ubiquitinated proteins are reduced in WASH − / − MEFs . WASH + / + and WASH − / − MEFs were starved in EBSS for 2 h treated with or without 20 nM BafA1 or 10 μM MG132 . Cells were harvested for immunoblotting with anti ‐ poly ‐ ubiquitin antibody ( Enzo , clone FK1 ) that only recognizes poly ‐ ubiquitinated ubiquitin chains .

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

(C) Poly‐ubiquitinated proteins are reduced in WASH−/− MEFs. WASH+/+ and WASH−/− MEFs were starved in EBSS for 2 h treated with or without 20 nM BafA1 or 10 μM MG132. Cells were harvested for immunoblotting with anti‐poly‐ubiquitin antibody (Enzo, clone FK1) that only recognizes poly‐ubiquitinated ubiquitin chains.


( D ) Autophagosome ‐ like structures in WASH + / + and WASH − / − MEFs were visualized after starvation for 1 h by immuno ‐ electron microscopy with antibody against LC3 ( left panel ) . Black arrowhead indicates autophagosome . Scale bar , 500 nm . * , Cellular reference . Fifty cells were quantified from three independent experiments ( right panel ) . Data were shown as means ± s . d . * P0 . 05 and * * P0 . 01 . Experiments were repeated for three independent times with similar results . Source data for this figure is available on the online supplementary information page .

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 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

(D) Autophagosome‐like structures in WASH+/+ and WASH−/− MEFs were visualized after starvation for 1 h by immuno‐electron microscopy with antibody against LC3 (left panel). Black arrowhead indicates autophagosome. Scale bar, 500 nm. *, Cellular reference. Fifty cells were quantified from three independent experiments (right panel). Data were shown as means±s.d. *P0.05 and **P0.01. Experiments were repeated for three independent times with similar results.Source data for this figure is available on the online supplementary information page.


( A ) WASH colocalizes with GFP ‐ LC3 upon starvation . HeLa cells stably expressing GFP ‐ LC3 were stained with anti ‐ WASH antibody after treatment with EBSS or culture medium ( CM ) for 1 h .

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

(A) WASH colocalizes with GFP‐LC3 upon starvation. HeLa cells stably expressing GFP‐LC3 were stained with anti‐WASH antibody after treatment with EBSS or culture medium (CM) for 1 h.


( B ) Autophagy ‐ related WASH does not colocalize with EEA1 . HeLa cells stably expressing GFP ‐ LC3 were cultured with EBSS for 1 h and stained with antibodies against EEA1 and WASH .

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

(B) Autophagy‐related WASH does not colocalize with EEA1. HeLa cells stably expressing GFP‐LC3 were cultured with EBSS for 1 h and stained with antibodies against EEA1 and WASH.


( C ) WASH localizes to unclosed autophagosomes . HeLa cells stably expressing GFP ‐ Atg5 were transfected with Atg4B ( C74A ) mutant , followed by stimulation with EBSS for 1 h . Cells were stained with anti ‐ WASH antibody ( red ) .

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

(C) WASH localizes to unclosed autophagosomes. HeLa cells stably expressing GFP‐Atg5 were transfected with Atg4B(C74A) mutant, followed by stimulation with EBSS for 1 h. Cells were stained with anti‐WASH antibody (red).


( D ) WASH does not localize in autolysosomes . HeLa cells were treated with CM or 1 h in the presence or absence of 20 nM BafA1 at 37 ° C for 1 h . WASH and LAMP1 were visualized by staining with anti ‐ WASH and anti ‐ LAMP1 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 B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O O

(D) WASH does not localize in autolysosomes. HeLa cells were treated with CM or 1 h in the presence or absence of 20 nM BafA1 at 37°C for 1 h. WASH and LAMP1 were visualized by staining with anti‐WASH and anti‐LAMP1 antibodies.


( E ) Colocalization of WASH and autophagy substrate p62 during autophagy . HeLa cells treated with or without EBSS were stained with anti ‐ p62 and anti ‐ WASH antibodies . For ( A - E ) , scale bar , 10 μm .

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

(E) Colocalization of WASH and autophagy substrate p62 during autophagy. HeLa cells treated with or without EBSS were stained with anti‐p62 and anti‐WASH antibodies. For (A-E), scale bar, 10 μm.


( F ) Immuno ‐ electron microscopy analysis of EBSS cells treated with EBSS for 1 h . Black arrowhead indicates WASH particle , red arrowhead indicates autophagosome , and black arrow denotes autolysosome . In all , 73 ± 2 % of autophagosomes contains WASH particles . Scale bar , 100 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

(F) Immuno‐electron microscopy analysis of EBSS cells treated with EBSS for 1 h. Black arrowhead indicates WASH particle, red arrowhead indicates autophagosome, and black arrow denotes autolysosome. In all, 73±2% of autophagosomes contains WASH particles. Scale bar, 100 nm.


( G ) WASH is not localized in autolysosomes . Autolysosomes and early endosomes were separated from HeLa cells and immunoblotted with indicated antibodies . The above experiments were repeated for three independent times with similar results . Source data for this figure is available on the online supplementary information page .

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

(G) WASH is not localized in autolysosomes. Autolysosomes and early endosomes were separated from HeLa cells and immunoblotted with indicated antibodies. The above experiments were repeated for three independent times with similar results.Source data for this figure is available on the online supplementary information page.


( A ) WASH deficiency enhances the interaction of Vps34 with Beclin 1 . WASH + / + and WASH − / − MEFs were treated with EBSS at 37 ° C for 1 h and immunoprecipitated for Beclin 1 . IP , immunoprecipitation . Ratio of Vps34 / Beclin 1 was calculated by the Image J software and shown in the right panel .

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

(A) WASH deficiency enhances the interaction of Vps34 with Beclin 1. WASH+/+ and WASH−/− MEFs were treated with EBSS at 37°C for 1 h and immunoprecipitated for Beclin 1. IP, immunoprecipitation. Ratio of Vps34/Beclin 1 was calculated by the Image J software and shown in the right panel.


( B ) WASH KO MEFs were treated with or without EBSS at 37 ° C for EBSS followed by kinase assays . Cells were lysed and incubated with anti ‐ Beclin 1 antibody to precipitate the autophagy ‐ related Vps34 . Immunoprecipitates were separated into two equal parts , one for kinase assays and the other for input detection . RLU , relative light unit .

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-MEASURED_VAR I-MEASURED_VAR 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

(B) WASH KO MEFs were treated with or without EBSS at 37°C for EBSS followed by kinase assays. Cells were lysed and incubated with anti‐Beclin 1 antibody to precipitate the autophagy‐related Vps34. Immunoprecipitates were separated into two equal parts, one for kinase assays and the other for input detection. RLU, relative light unit.


( C ) WASH deficiency enhances Vps34 activity . WASH + / + and WASH − / − MEFs stably expressing GFP ‐ WIPI1 were stimulated with or without EBSS for 1 h followed by confocal microscopy ( left panel ) . GFP ‐ WIPI1 dots were calculated and shown in the right panel . Scale bar , 10 μm .

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

(C) WASH deficiency enhances Vps34 activity. WASH+/+ and WASH−/− MEFs stably expressing GFP‐WIPI1 were stimulated with or without EBSS for 1 h followed by confocal microscopy (left panel). GFP‐WIPI1 dots were calculated and shown in the right panel. Scale bar, 10 μm.


( D ) WASH ‐ silenced HeLa cells were treated with or without EBSS at 37 ° C for 1 h , and detected as above .

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

(D) WASH‐silenced HeLa cells were treated with or without EBSS at 37°C for 1 h, and detected as above.


( E ) WASH attenuates the interaction of Beclin 1 with Vps34 . MEFs stably expressing vector or Flag - WASH were starved with EBSS for 1 h , followed by immunoprecipitation with anti ‐ Beclin 1 antibody . Ratio of Vps34 / Beclin 1 was shown in the right panel .

O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR 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 B-MEASURED_VAR I-MEASURED_VAR O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O

(E) WASH attenuates the interaction of Beclin 1 with Vps34. MEFs stably expressing vector or Flag-WASH were starved with EBSS for 1 h, followed by immunoprecipitation with anti‐Beclin 1 antibody. Ratio of Vps34/Beclin 1 was shown in the right panel.


( F ) WASH overexpression reduces Vps34 activity . MEFs stably expressing GFP ‐ WIPI1 were transfected with vector or Flag - WASH and then starved with EBSS for 1 h , followed by visualization with confocal microscopy ( upper panel ) . GFP ‐ WIPI1 dots were calculated and shown in the lower panel . Scale bar , 10 μm . Data are shown as means ± s . d . * * P0 . 01 and * * * P0 . 001 . Experiments were repeated for three independent times with similar results . Source data for this figure is available on the online supplementary information page .

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

(F) WASH overexpression reduces Vps34 activity. MEFs stably expressing GFP‐WIPI1 were transfected with vector or Flag-WASH and then starved with EBSS for 1 h, followed by visualization with confocal microscopy (upper panel). GFP‐WIPI1 dots were calculated and shown in the lower panel. Scale bar, 10 μm. Data are shown as means±s.d. **P0.01 and ***P0.001. Experiments were repeated for three independent times with similar results.Source data for this figure is available on the online supplementary information page.


( A ) The interaction of WASH with Beclin 1 was validated by yeast two ‐ hybrid assays . Yeast strain AH109 was co ‐ transfected with Gal4 DNA ‐ binding domain ( BD ) fused WASH and Gal4 activating domain ( AD ) fused Beclin 1 . p53 and large T antigen or a known WASH interactor Bloc1s2 was introduced as positive controls .

O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O

(A) The interaction of WASH with Beclin 1 was validated by yeast two‐hybrid assays. Yeast strain AH109 was co‐transfected with Gal4 DNA‐binding domain (BD) fused WASH and Gal4 activating domain (AD) fused Beclin 1. p53 and large T antigen or a known WASH interactor Bloc1s2 was introduced as positive controls.


( B , C ) WASH co ‐ immunoprecipitates Beclin 1 in mammalian cells . Flag ‐ tagged Beclin 1 and Myc ‐ tagged WASH were co ‐ transfected into HEK293T cells and immunoprecipitations were performed at 24 h post transfection . IP , immunoprecipitation .

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

(B, C) WASH co‐immunoprecipitates Beclin 1 in mammalian cells. Flag‐tagged Beclin 1 and Myc‐tagged WASH were co‐transfected into HEK293T cells and immunoprecipitations were performed at 24 h post transfection. IP, immunoprecipitation.


( D ) Mapping the interacting regions between WASH and Beclin 1 .

O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O

(D) Mapping the interacting regions between WASH and Beclin 1.


( E ) The indicated Flag ‐ tagged Beclin 1 constructs encoding different regions of Beclin 1 were co ‐ transfected with full ‐ length HA ‐ tagged WASH ( HA - WASH ) into HEK293T cells followed by immunoprecipitation .

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

(E) The indicated Flag‐tagged Beclin 1 constructs encoding different regions of Beclin 1 were co‐transfected with full‐length HA‐tagged WASH (HA-WASH) into HEK293T cells followed by immunoprecipitation.


( F ) WASH directly interacts with Beclin 1 . Recombinant MBP ‐ tagged WASH ( MBP - WASH ) or Beclin 1 ( rBeclin 1 ) was expressed in E . coli and then subjected to MBP pull ‐ down assay .

O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O

(F) WASH directly interacts with Beclin 1. Recombinant MBP‐tagged WASH (MBP-WASH) or Beclin 1 (rBeclin 1) was expressed in E. coli and then subjected to MBP pull‐down assay.


( G ) GST - Beclin 1 precipitates WASH from HeLa cell lysates . GST ‐ Beclin 1 or GST was bound to GST beads and incubated with HeLa cell lysates followed by immunoblotting .

O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O

(G) GST-Beclin 1 precipitates WASH from HeLa cell lysates. GST‐Beclin 1 or GST was bound to GST beads and incubated with HeLa cell lysates followed by immunoblotting.


( H ) Endogenous WASH colocalizes with Beclin 1 via confocal microscopy . HeLa cells cultured in CM or EBSS for 1 h were stained with antibodies against Beclin 1 and WASH . The colocalization rate ( Pearson ' s correlation coefficient ) between Beclin 1 and WASH was calculated and shown as means ± s . d . in the lower panel . Scale bar , 10 μm .

O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O

(H) Endogenous WASH colocalizes with Beclin 1 via confocal microscopy. HeLa cells cultured in CM or EBSS for 1 h were stained with antibodies against Beclin 1 and WASH. The colocalization rate (Pearson's correlation coefficient) between Beclin 1 and WASH was calculated and shown as means±s.d. in the lower panel. Scale bar, 10 μm.


( I ) HeLa cells were treated with CM or EBSS for 1 h , followed by immunoprecipitation with antibodies against WASH ( left panel ) or Beclin 1 ( right panel ) , and the immunoprecipitates were detected with the indicated antibodies . * * * P0 . 001 . All the above experiments were repeated for at least three times with similar results . Source data for this figure is available on the online supplementary information page .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(I) HeLa cells were treated with CM or EBSS for 1 h, followed by immunoprecipitation with antibodies against WASH (left panel) or Beclin 1 (right panel), and the immunoprecipitates were detected with the indicated antibodies.***P0.001. All the above experiments were repeated for at least three times with similar results.Source data for this figure is available on the online supplementary information page.


( A ) Endogenous Beclin 1 is ubiquitinated through K63 linkage during the process of autophagy . EBSS cells starved in EBSS for the indicated times were lysed and immunoprecipitated with anti ‐ Beclin 1 antibody . Immunoprecipitates were dissociated with 1 % SDS and re ‐ immunoprecipitated with anti ‐ Beclin 1 antibody , followed by immunoblotting with antibody specific for lysine 63 ‐ linked poly ‐ ubiquitin chains ( anti ‐ K63 ‐ Ub ) . (

O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O

(A) Endogenous Beclin 1 is ubiquitinated through K63 linkage during the process of autophagy. EBSS cells starved in EBSS for the indicated times were lysed and immunoprecipitated with anti‐Beclin 1 antibody. Immunoprecipitates were dissociated with 1% SDS and re‐immunoprecipitated with anti‐Beclin 1 antibody, followed by immunoblotting with antibody specific for lysine 63‐linked poly‐ubiquitin chains (anti‐K63‐Ub). (


( B ) The autophagy ‐ specific Beclin 1 is ubiquitinated through K63 linkage . After starved in EBSS for 1 h , HeLa cells were lysed and HeLa with anti ‐ Atg14L antibody before dissociated with 1 % SDS . Immunoprecipitates were further precipitated with anti ‐ Beclin 1 antibody , followed by immunoblotting with anti ‐ K63 ‐ Ub antibody . The left panel denotes the scheme for separation of autophagy ‐ specific Beclin 1 . The right panel indicates ubiquitination state of autophagy ‐ specific Beclin 1 .

O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O

(B) The autophagy‐specific Beclin 1 is ubiquitinated through K63 linkage. After starved in EBSS for 1 h, HeLa cells were lysed and HeLa with anti‐Atg14L antibody before dissociated with 1% SDS. Immunoprecipitates were further precipitated with anti‐Beclin 1 antibody, followed by immunoblotting with anti‐K63‐Ub antibody. The left panel denotes the scheme for separation of autophagy‐specific Beclin 1. The right panel indicates ubiquitination state of autophagy‐specific Beclin 1.


( C ) Lysine 437 mutation to arginine ( K437R ) abrogates ubiquitination of Beclin 1 . Different constructs of Flag ‐ tagged Beclin 1 mutants were co ‐ transfected with HA ‐ tagged ubiquitin ( HA - Ub ) into HEK293T cells .

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

(C) Lysine 437 mutation to arginine (K437R) abrogates ubiquitination of Beclin 1. Different constructs of Flag‐tagged Beclin 1 mutants were co‐transfected with HA‐tagged ubiquitin (HA-Ub) into HEK293T cells.


( D ) Beclin 1 ( WT ) or K437R ‐ Beclin 1 ( K437R ) mutant was stably expressed in Beclin 1 ‐ silenced HeLa cells . Beclin 1 ‐ knockdown HeLa cells were infected with lentivirus encoding 3XFlag ‐ tagged WT Beclin 1 ( WT ) or K437R ‐ Beclin 1 ( K437R ) .

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

(D) Beclin 1 (WT) or K437R‐Beclin 1 (K437R) mutant was stably expressed in Beclin 1‐silenced HeLa cells. Beclin 1‐knockdown HeLa cells were infected with lentivirus encoding 3XFlag‐tagged WT Beclin 1 (WT) or K437R‐Beclin 1 (K437R).


( E ) K437R ‐ Beclin 1 mutant abrogates the enhanced interaction of Beclin 1 with Vps34 during autophagy . HeLa cells expressing 3XFlag - Beclin 1 or 3XFlag - K437R ‐ Beclin 1 were treated with EBSS for 1 h , and harvested for immunoprecipitation .

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

(E) K437R‐Beclin 1 mutant abrogates the enhanced interaction of Beclin 1 with Vps34 during autophagy. HeLa cells expressing 3XFlag-Beclin 1 or 3XFlag-K437R‐Beclin 1 were treated with EBSS for 1 h, and harvested for immunoprecipitation.


( F ) Vps34 kinase activity is impaired by K437R ‐ Beclin 1 mutant . HeLa cells were transfected with the indicated vectors for 24 h and immunoprecipitated with anti ‐ HA antibody followed by a Vps34 kinase assay . Immunoprecipitates were separated into two equal parts , one for kinase assays and the other for input detection .

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

(F) Vps34 kinase activity is impaired by K437R‐Beclin 1 mutant. HeLa cells were transfected with the indicated vectors for 24 h and immunoprecipitated with anti‐HA antibody followed by a Vps34 kinase assay. Immunoprecipitates were separated into two equal parts, one for kinase assays and the other for input detection.


( G ) K437R ‐ Beclin 1 reduces the activation of Vps34 . Beclin 1 ‐ silenced HeLa cells stably expressing GFP ‐ HeLa were rescued with WT ‐ or K437R ‐ Beclin 1 , followed by stimulation with 1 h for 1 h . GFP ‐ WIPI1 dots were visualized by confocal microscopy .

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

(G) K437R‐Beclin 1 reduces the activation of Vps34. Beclin 1‐silenced HeLa cells stably expressing GFP‐HeLa were rescued with WT‐ or K437R‐Beclin 1, followed by stimulation with 1 h for 1 h. GFP‐WIPI1 dots were visualized by confocal microscopy.


( H ) K437R ‐ Beclin 1 mutant fails to induce autophagy after starvation . Beclin 1 ‐ knockdown HeLa cells expressing WT or K437R mutant of Beclin 1 ( K437R ) were starved with EBSS for the indicated times .

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

(H) K437R‐Beclin 1 mutant fails to induce autophagy after starvation. Beclin 1‐knockdown HeLa cells expressing WT or K437R mutant of Beclin 1 (K437R) were starved with EBSS for the indicated times.


( I ) K437R ‐ Beclin 1 mutant suppresses autophagosome formation . Beclin 1 ‐ knockdown HeLa cells expressing WT ‐ Beclin 1 or K437R ‐ Beclin 1 were infected with lentivirus encoding GFP ‐ LC3 , and stimulated with EBSS for 1 h in the presence or absence of 20 nM BafA1 . GFP ‐ LC3 dots were visualized by confocal microscopy . Scale bar , 10 μm . Data are shown as means ± s . d . * * P0 . 01 and * * * P0 . 001 . All the above experiments were repeated for at least three times with similar results . Source data for this figure is available on the online supplementary information page .

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

(I) K437R‐Beclin 1 mutant suppresses autophagosome formation. Beclin 1‐knockdown HeLa cells expressing WT‐Beclin 1 or K437R‐Beclin 1 were infected with lentivirus encoding GFP‐LC3, and stimulated with EBSS for 1 h in the presence or absence of 20 nM BafA1. GFP‐LC3 dots were visualized by confocal microscopy. Scale bar, 10 μm. Data are shown as means±s.d. **P0.01 and ***P0.001. All the above experiments were repeated for at least three times with similar results.Source data for this figure is available on the online supplementary information page.


( A ) WASH overexpression suppresses the ubiquitination of Beclin 1 . HEK293T cells were transfected with the indicated vectors for 24 h followed by an Ni ‐ NTA ‐ based pull ‐ down assay and immunoblotted with anti ‐ poly ‐ ubiquitin ( anti ‐ poly ‐ Ub ) ( left panel ) , and anti ‐ K63 ‐ specific ubiquitin ( anti ‐ K63 ‐ Ub ) ( middle panel ) antibodies . The same blot was stripped and probed with anti ‐ K48 ‐ specific ubiquitin ( anti ‐ K48 ‐ Ub ) antibody ( right panel ) .

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

(A) WASH overexpression suppresses the ubiquitination of Beclin 1. HEK293T cells were transfected with the indicated vectors for 24 h followed by an Ni‐NTA‐based pull‐down assay and immunoblotted with anti‐poly‐ubiquitin (anti‐poly‐Ub) (left panel), and anti‐K63‐specific ubiquitin (anti‐K63‐Ub) (middle panel) antibodies. The same blot was stripped and probed with anti‐K48‐specific ubiquitin (anti‐K48‐Ub) antibody (right panel).


( B ) WASH overexpression hinders K63 ‐ linked ubiquitination of Beclin 1 . HeLa cells stably expressing vector or Flag - WASH were treated with CM or EBSS with or without BafA1 for 1 h and then stained with antibodies against endogenous Beclin 1 and K63 ‐ linked polyubiquitin ( K63 ‐ Ub ) . The colocalization rate ( Pearson ' s correlation coefficient ) between Beclin 1 and K63 ‐ Ub was calculated and shown in the right panel .

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

(B) WASH overexpression hinders K63‐linked ubiquitination of Beclin 1. HeLa cells stably expressing vector or Flag-WASH were treated with CM or EBSS with or without BafA1 for 1 h and then stained with antibodies against endogenous Beclin 1 and K63‐linked polyubiquitin (K63‐Ub). The colocalization rate (Pearson's correlation coefficient) between Beclin 1 and K63‐Ub was calculated and shown in the right panel.


( C ) WASH overexpression inhibits LC3 lipidation . HeLa cells stably expressing vector or Flag - WASH were treated with CM or EBSS with or without BafA1 for 1 h and then stained with antibody against endogenous LC3 . LC3 dots were calculated and shown in the right panel .

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

(C) WASH overexpression inhibits LC3 lipidation. HeLa cells stably expressing vector or Flag-WASH were treated with CM or EBSS with or without BafA1 for 1 h and then stained with antibody against endogenous LC3. LC3 dots were calculated and shown in the right panel.


( D ) WASH deficiency increases K63 ‐ linked ubiquitination of Beclin 1 . WASH + / + and WASH − / − MEFs were treated with EBSS at 37 ° C for 1 h and lysed for immunoprecipitation ( IP ) with anti ‐ Beclin 1 antibody and probed with antibody against K63 ‐ linked poly ‐ ubiquitin .

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

(D) WASH deficiency increases K63‐linked ubiquitination of Beclin 1. WASH+/+ and WASH−/− MEFs were treated with EBSS at 37°C for 1 h and lysed for immunoprecipitation (IP) with anti‐Beclin 1 antibody and probed with antibody against K63‐linked poly‐ubiquitin.


( E ) WASH overexpression inhibits Vps34 activity . HeLa cells were transfected with the indicated vectors and subjected to immunoprecipitation with anti ‐ Beclin 1 antibody for Vps34 kinase activity assay . Immunoprecipitates were separated into two equal parts , one for kinase assay and the other for input detection . RLU , relative light unit .

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

(E) WASH overexpression inhibits Vps34 activity. HeLa cells were transfected with the indicated vectors and subjected to immunoprecipitation with anti‐Beclin 1 antibody for Vps34 kinase activity assay. Immunoprecipitates were separated into two equal parts, one for kinase assay and the other for input detection. RLU, relative light unit.


( F ) Beclin 1 associates Ambra1 upon autophagy induction . HeLa cells treated with EBSS for 1 h were lysed for immunoprecipitation with anti ‐ Beclin 1 antibody . Immunoprecipitates were immunoblotted with the indicated antibodies .

O O O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O

(F) Beclin 1 associates Ambra1 upon autophagy induction. HeLa cells treated with EBSS for 1 h were lysed for immunoprecipitation with anti‐Beclin 1 antibody. Immunoprecipitates were immunoblotted with the indicated antibodies.