G showing top five active regulons in each cluster , on expression data of multi - omics dataset . TF regulons also identified by other approaches are highlighted .

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G Heatmap showing the top five active regulons in each cell cluster, based on the expression data of the multi-omics dataset. TF regulons also identified by other approaches are highlighted.


A Average histone modification profiles of undifferentiated differentiated keratinocyte - specific peaks using previously published data generated on NHEK ( ENCODE ) . normalized signal of H3K27ac , H3K4me1 / me2 / me3 H3K27me3 were measured in a window of ± 10 , 000 base - pair ( bp ) .

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A Average histone modification profiles of undifferentiated and differentiated keratinocyte-specific peaks using previously published data generated on NHEK cells (ENCODE). The normalized signal of H3K27ac, H3K4me1/me2/me3 and H3K27me3 were measured in a window of ±10,000 base-pair (bp).


B Fractions of CpGs located within epigenomic substructures for 4 , 351 InfiniumEPIC CpG probes found within undifferentiated differentiated keratinocyte - specific peaks .

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B Fractions of CpGs located within epigenomic substructures for the 4,351 InfiniumEPIC CpG probes found within undifferentiated and differentiated keratinocyte-specific peaks.


C Unsupervised hierarchical clustering of 12 healthy , 20 AK 35 cSCC epidermal samples on methylation status at undifferentiated differentiated keratinocyte - specific peaks . Each row represents average methylation value of CpGs contained in a particular peak .

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C Unsupervised hierarchical clustering of 12 healthy, 20 AK and 35 cSCC epidermal samples based on the methylation status at undifferentiated and differentiated keratinocyte-specific peaks. Each row represents the average methylation value of all CpGs contained in a particular peak.


B , C Average methylation in 15 states defined by ChromHMM in NHEK for ( B ) both EpSC Keratinocytes from sci - MET single - dataset , or for ( C ) EpSC - like keratinocyte - like AK / cSCC from EPIC dataset . ChromHMM state 1 : active promoter ; state 2 : weak promoter ; state 3 : inactive / poised promoter ; state 4 : strong enhancer ; state 5 : strong enhancer ; state 6 : weak / poised enhancer ; state 7 : weak / poised enhancer ; state 8 : insulator ; state 9 : transcriptional transition ; state 10 : transcriptional elongation ; state 11 : weak transcription ; state 12 : polycomb - repressed ; state 13 : heterochromatin / low signal ; state 14 : repetitive / copy number variation ; state 15 : repetitive / copy number variation . AK : actinic keratosis , cSCC : cutaneous squamous carcinoma . Data information : Statistical analyses in B C were performed using a Wilcoxon Rank Sum comparing average methylation values in genomic states 4 5 between EpSC ( n = 6 ) keratinocytes ( n = 548 ) ( B ) or EpSC - like ( n = 22 ) keratinocyte - like ( n = 33 ) AK / cSCC samples ( C ) . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

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B, C Average DNA methylation in the 15 chromatin states defined by ChromHMM in NHEK for (B) both EpSC and Keratinocytes from the sci-MET single-cell dataset, or for (C) EpSC-like and keratinocyte-like AK/cSCC from the EPIC dataset. ChromHMM state 1: active promoter; state 2: weak promoter; state 3: inactive/poised promoter; state 4: strong enhancer; state 5: strong enhancer; state 6: weak/poised enhancer; state 7: weak/poised enhancer; state 8: insulator; state 9: transcriptional transition; state 10: transcriptional elongation; state 11: weak transcription; state 12: polycomb-repressed; state 13: heterochromatin/low signal; state 14: repetitive/copy number variation; state 15: repetitive/copy number variation. AK: actinic keratosis, cSCC: cutaneous squamous cell carcinoma. Data information: Statistical analyses in B and C were performed using a Wilcoxon Rank Sum test comparing the average methylation values in genomic states 4 and 5 between EpSC (n= 6) and keratinocytes (n= 548) (B) or EpSC-like (n= 22) and keratinocyte-like (n= 33) AK/cSCC samples (C). *P <0.05, **P <0.01, ***P <0.001, ****P<0.0001.


D Heatmaps displaying gene expression ( upper ) imputed promoter methylation ( lower ) reference matrices for keratinocyte populations involved in terminal differentiation , calculated by EpiSCORE . of each gene in reference matrix is also depicted .

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D Heatmaps displaying the gene expression (upper) and the imputed promoter DNA methylation (lower) reference matrices for keratinocyte populations involved in terminal differentiation, calculated by EpiSCORE. The weight of each gene in the reference DNA matrix is also depicted.


C Upper : Heatmaps displaying unsupervised clustering of 67 AK / cSCC healthy controls on methylation patterns of MIR200C / 141 cluster MIR205 gene . Lower : Bar plots showing of methylation values at CpGs located at promoter regions in each - of - origin - subclass . For MIR200C / 141 cluster , probes located at regulatory CpG island are depicted with green arrows . For MIR205 gene , probes shown in are part of promoter region were used for . Data information : AK : actinic keratosis , cSCC : cutaneous squamous carcinoma .

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C Upper: Heatmaps displaying the unsupervised clustering of 67 AK/cSCC and healthy controls based on the methylation patterns of the MIR200C/141 cluster and the MIR205 gene. Lower: Bar plots showing the quantification of the methylation values at CpGs located at promoter regions in each cell-of-origin-based subclass. For the MIR200C/141 cluster, probes located at the regulatory CpG island are depicted with green arrows. For the MIR205 gene, all probes shown in the heatmap are part of the promoter region and were used for quantification. Data information: AK: actinic keratosis, cSCC: cutaneous squamous cell carcinoma.


D Left : Representative of EpSC - like ( n = 5 ) Keratinocyte - like ( n = 5 ) cSCC tumors stained for EMT - marker ZEB2 ( green ) keratinocyte marker TP63 ( red ) . Nuclei were counterstained with DAPI . are shown at 40x original magnification . Scale bar , 50 µm . Right : of ZEB2 - positive ( TP63 - positive ) in three independent regions per sample . Each dot represents a region with at least 500 ( TP63 - positive ) counted . Data information : Statistical in D was performed using an unpaired two - sided t - ( EpSC - like : n = 5 samples , 15 technical replicates ; keratinocyte - like : n = 5 samples , 15 technical replicates ) . Central bar represents mean error bars represent standard deviation . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

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D Left: Representative images of EpSC-like (n=5) and Keratinocyte-like (n=5) cSCC tumors stained for the EMT-marker ZEB2 (green) and the keratinocyte marker TP63 (red). Nuclei were counterstained with DAPI. Images are shown at 40x original magnification. Scale bar, 50 µm. Right: Quantification of ZEB2-positive tumor cells (TP63-positive) in three independent regions per sample. Each dot represents a region with at least 500 tumor cells (TP63-positive) counted. Data information: Statistical analysis in D was performed using an unpaired two-sided t-test (EpSC-like: n= 5 samples, 15 technical replicates; keratinocyte-like: n= 5 samples, 15 technical replicates). Central bar represents the mean and error bars represent the standard deviation. *P <0.05, **P <0.01, ***P <0.001, ****P<0.0001.


( D , E ) Representative examples of dose - response curves time courses . Lines through dose - response curves are fits to Michaelis - Menten equation ; time courses are fitted to linear equations . Shown are means standard errors over at least three independent repeats .

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(D, E) Representative examples of dose-response curves and time courses. Lines through the dose-response curves are fits to the Michaelis-Menten equation; time courses are fitted to linear equations. Shown are means and standard errors over at least three independent repeats.


( F ) Fold increase ( positive values ) or decrease ( negative values ) in initial uptake rates of variants relative to WT GltPh . Circles are averages of technical triplicates . Bars are means over independent repeats ( as indicated ) . dashed marks a four - fold increase in initial rate .

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(F) Fold increase (positive values) or decrease (negative values) in the initial uptake rates of the variants relative to WT GltPh. Circles are averages of technical triplicates. Bars are means over independent repeats (as indicated). The dashed line marks a four-fold increase in the initial rate.


( A , B ) Schematic representations of experimental conditions are shown on far left . Data are shown for WT GltPh select gain - of - function mutants , as indicated above panels . Transition density plots ( top of each panel ) show frequency of transitions between EFRET values . number of trajectories analyzed ( N ) population - wide mean frequency of transitions is shown on panels . Representative 50 s sections of single - molecule EFRET trajectories ( middle ) with raw data in black idealizations in brown . Scale bar conformational states corresponding to low intermediate EFRET values are shown to left of panels . Dwell - time distributions for OFS in blue IFS in red ( bottom ) . Mean dwell times are shown on panels in corresponding colors . Black lines represent photobleaching survival plots normalized from 1 to 0 . Shown are means standard errors over at least three independent repeats .

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(A, B) Schematic representations of the experimental conditions are shown on the far left. Data are shown for WT GltPh and select gain-of-function mutants, as indicated above the panels. Transition density plots (top of each panel) show the frequency of transitions between the EFRET values. The number of trajectories analyzed (N) and the population-wide mean frequency of transitions is shown on the panels. Representative 50 s sections of single-molecule EFRET trajectories (middle) with raw data in black and idealizations in brown. Scale bar and the conformational states corresponding to the low and intermediate EFRET values are shown to the left of the panels. Dwell-time distributions for the OFS in blue and the IFS in red (bottom). Mean dwell times are shown on the panels in corresponding colors. Black lines represent photobleaching survival plots normalized from 1 to 0. Shown are means and standard errors over at least three independent repeats.


( A ) Changes in initial rates of substrate uptake ( gray bars ) , transport domain dynamics ( black bars ) , l - Asp dissociation constant ( bars ) of mutant GltPh variants relative to WT transporter . transition frequencies were measured under non - equilibrium transport conditions , frequencies obtained in presence of d , l - TBOA were subtracted from data . Error bars represent standard errors for at least three independent measurements .

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(A) Changes in the initial rates of substrate uptake (gray bars), transport domain dynamics (black bars), and l-Asp dissociation constant (white bars) of the mutant GltPh variants relative to the WT transporter. The transition frequencies were measured under non-equilibrium transport conditions, and frequencies obtained in the presence of d,l-TBOA were subtracted from the data. Error bars represent standard errors for at least three independent measurements.


( A ) Dwell - time distributions of OFS ( blue ) IFS ( red ) for WT ( top ) K290A ( bottom ) GltPh observed under equilibrium conditions in saturating Na + / l - Asp ( left ) . Lines are fits to three exponentials . Data are averages standard errors of at least three independent measurements .

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(A) Dwell-time distributions of the OFS (blue) and the IFS (red) for WT (top) and K290A (bottom) GltPh observed under equilibrium conditions in saturating Na+/l-Asp (left). Lines are fits to three exponentials. Data are averages and standard errors of at least three independent measurements.


( B ) Representative EFRET trajectories of WT GltPh showing different transition frequencies . Raw data are in black , idealizations are in brown . Scalebar is above trajectories .

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(B) Representative EFRET trajectories of WT GltPh showing different transition frequencies. Raw data are in black, and idealizations are in brown. Scalebar is above the trajectories.


( C ) Distribution of WT ( top ) K290A GltPh ( bottom ) molecules with different mean transition frequencies ( bars ) . N is number of trajectories longer than 90 s used in . stacked black bars are fractions of trajectories without transitions . Pink bars show expected binomial distribution if trajectories shared mean transition frequency of 0 . 04 s - 1 for WT 0 . 34 s - 1 for K290A . Data are averages standard errors of at least three independent measurements .

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(C) Distribution of the WT (top) and the K290A GltPh (bottom) molecules with different mean transition frequencies (white bars). N is the number of trajectories longer than 90 s used in the analysis. The stacked black bars are fractions of trajectories without transitions. Pink bars show the expected binomial distribution if all trajectories shared the mean transition frequency of 0.04 s-1 for the WT and 0.34 s-1 for the K290A. Data are averages and standard errors of at least three independent measurements.


( D ) 2D - histograms of consecutive dwell lengths in OFS ( top ) IFS ( bottom ) . From left to right : calculated distribution of dwell times randomly selected from distributions in panel A ; measured distribution for WT GltPh K290A mutant .

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(D) 2D-histograms of the consecutive dwell lengths in the OFS (top) and the IFS (bottom). From left to right: calculated distribution of dwell times randomly selected from the distributions in panel A; measured distribution for WT GltPh and the K290A mutant.


( E ) Representative trajectories of WT ( left ) K290A ( right ) GltPh molecules showing switching between dynamic modes . Raw data are in black idealizations are in brown ( top ) . Black grey bars under trajectories indicate apparent slower faster dynamic modes . Survival plots for OFS IFS ( middle ) . Solid lines are fits to a single ( IFS ) double ( OFS ) exponentials . Dashed lines ( OFS only ) are rejected fits to single exponentials . Autocorrelation plots of sequential dwell durations ( bottom ) .

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(E) Representative trajectories of the WT (left) and the K290A (right) GltPh molecules showing switching between dynamic modes. Raw data are in black and idealizations are in brown (top). Black and grey bars under the trajectories indicate apparent slower and faster dynamic modes. Survival plots for the OFS and the IFS (middle). Solid lines are fits to a single (IFS) and double (OFS) exponentials. Dashed lines (OFS only) are rejected fits to single exponentials. Autocorrelation plots of the sequential dwell durations (bottom).


( A , B ) 2D - histograms of lifetime pairs obtained for trajectories exhibiting single exponential behavior . N is number of traces used . Scale bar shows relative density normalized by number of molecules . Above to right of each panel are stacked histograms of , respectively , OFS IFS lifetimes of analyzed molecules ( open bars ) photobleaching time of molecules showing no transitions ( black bars ) . Data from three independent measurements were combined for presentation . ( C ) mean lifetimes of molecules falling within 30 % of most populated bins of 2D - histograms ( yellow to red ) . open blue bar represents calculated mean OFS lifetime for WT GltPh Shown are means standard errors of mean of at least three independent measurements .

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(A, B) 2D-histograms of lifetime pairs obtained for trajectories exhibiting single exponential behavior. N is the number of traces used. Scale bar shows relative density normalized by the number of molecules. Above and to the right of each panel are stacked histograms of, respectively, the OFS and the IFS lifetimes of the analyzed molecules (open bars) and the photobleaching time of molecules showing no transitions (black bars). Data from three independent measurements were combined for presentation. (C) The mean lifetimes of the molecules falling within 30 % of the most populated bins of the 2D-histograms (yellow to red). The open blue bar represents the calculated mean OFS lifetime for WT GltPh Shown are means and standard errors of the mean of at least three independent measurements.


A , B Temporal expression of Snail genes in intestinal tract in ( A ) embryonic E12 . 5 to E18 . 5 ( B ) postnatal P0 to P20 measured by qRT - PCR . Shown are means ± SD .

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A, B Temporal expression of Snail genes in the intestinal tract in (A) embryonic E12.5 to E18.5 and (B) postnatal P0 to P20 measured by qRT-PCR. Shown are means ± SD.


C , E , G , I Immunohistochemical for Snai1 / 2 ( Abcam ab85931 ) ( C ) E14 . 5midgut , ( E ) E14 . 5hind gut , ( G ) postnatal day 14 SI , ( I ) postnatal day 14 colon . Snai1immunohistochemistry peptide competition controls are shown as inserts ( C ' , E ' , G ' , I ' ) . Scale bars : 20 mm . D , F , H , J , K X ‐ gal for β ‐ galactosidase activity in tissue from Snai2LacZ mice stained in whole mount , sectioned counterstained with fast red ( D ) E14 . 5midgut , ( F ) E14 . 5hind gut , ( H ) postnatal day 14 SI , ( J ) postnatal day 14 colon , ( K ) adult SI . Controls for X ‐ gal are shown as inserts ( D ' , F ' , H ' , J ' ) . L , M , N Double immunostaining of small intestinalcrypts for Snai1 / 2 ( red ) ( M ) Lgr5 ‐ GFP ( green ) ; ( N ) merge shows Snai1 is present in both CBCstem proliferating transit amplifying . Scale bars : 10 mm . O , P Immunohistochemistry for Snai1 using a different antibody ( Genetex GTX125918 ) shows minimal expression of Snai1 in villi . ( P ) Expression of Snai1 is detected at base of crypts ( P ) with strong expression in CBCstem indicated by arrows . Scale bars : 20 mm ( O ) , 10 mm ( P ) . Q Schematic diagram showing Snai1 expression ( red ) within different types in crypt . SI = small intestine . See also Supplementary Fig S1 .

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C, E, G, I Immunohistochemical staining for Snai1/2 (Abcam ab85931) (C) E14.5midgut, (E) E14.5hind gut, (G) postnatal day 14 SI, (I) postnatal day 14 colon. Snai1immunohistochemistry peptide competition controls are shown as inserts (C', E', G', I'). Scale bars: 20 mm.D, F, H, J, K X‐gal staining for β‐galactosidase activity in tissue from Snai2LacZ mice stained in whole mount, sectioned and counterstained with nuclear fast red (D) E14.5midgut, (F) E14.5hind gut, (H) postnatal day 14 SI, (J) postnatal day 14 colon, (K) adult SI. Controls for X‐gal staining are shown as inserts (D', F', H', J').L, M, N Double immunostaining of small intestinalcrypts for Snai1/2 (red) and (M) Lgr5‐GFP (green); (N) merge shows Snai1 is present in both CBCstem cells and proliferating transit amplifying cells. Scale bars: 10 mm.O, P Immunohistochemistry for Snai1 using a different primary antibody (Genetex GTX125918) shows minimal expression of Snai1 in villi. (P) Expression of Snai1 is detected at the base of crypts (P) with strong nuclear expression in CBCstem cells indicated by arrows. Scale bars: 20 mm (O), 10 mm (P).Q Schematic diagram showing Snai1 expression (red) within different cell types in the crypt. SI = small intestine. See also Supplementary Fig S1.


Periodic Acid Schiff ( PAS ) was used to detect goblet in sections of small intestine from control Snai1 KO mice . Graph shows number of goblet per crypt - villus axes ( n = 3 ) . Immunohistochemistry for synaptophysin was used to detect enteroendocrine ( marked by arrows ) . of enteroendocrine per crypt - villus axes in control Snai1 KO tissue revealed a significant increase in number of enteroendocrine following Snai1 loss ( n = 3 , P = 0 . 004 ) . Immunohistochemistry for lysozyme was used to detect Paneth . of Paneth in control Snai1 KO indicates a significant increase in Paneth numbers following knockout of Snai1 ( n = 4 , P = 0 . 002 ) .

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Periodic Acid Schiff (PAS) staining was used to detect goblet cells in sections of small intestine from control and Snai1 KO mice. Graph shows number of goblet cells per crypt-villus axes (n = 3).Immunohistochemistry for synaptophysin was used to detect enteroendocrine cells (marked by arrows). Quantification of enteroendocrine cells per crypt-villus axes in control and Snai1 KO tissue revealed a significant increase in the number of enteroendocrine cells following Snai1 loss (n = 3, P = 0.004).Immunohistochemistry for lysozyme was used to detect Paneth cells. Quantification of Paneth cells in control and Snai1 KO indicates a significant increase in Paneth cell numbers following knockout of Snai1 (n = 4, P = 0.002).


Measurement of villus ( μm ) ( n = 3 , P = 0 . 013 ) of enterocytes per crypt - villus unit demonstrate a significant reduction in absorptive enterocytes following Snai1 loss ( n = 3 , P = 0 . 036 ) . Measurement of villus ( μm ) ( n = 3 , P = 0 . 003 ) 8 days after β ‐ NF induction demonstrates a significant shortening of villi . Bars represent mean ± SD . Two ‐ Student ' s t ‐ was used to assess significance . Scale bars : 20 μm .

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Measurement of villus length (μm) (n = 3, P = 0.013) and quantification of enterocytes per crypt-villus unit demonstrate a significant reduction in absorptive enterocytes following Snai1 loss (n = 3, P = 0.036).Measurement of villus length (μm) (n = 3, P = 0.003) 8 days after β‐NF induction demonstrates a significant shortening of villi. Bars represent mean ± SD. Two‐tailed Student's t‐test was used to assess significance. Scale bars: 20 μm.


Sections from control Snai1 KO small intestine were stained for PCNA to detect proliferating ( arrows depicting proliferating CBC at base of crypts ) . of total PCNA ‐ positive per crypt PCNA ‐ positive in base of crypts both revealed a significant reduction in number of proliferating after conditional depletion of Snai1 ( n = 4 , P = 0 . 049 , 0 . 026 ) . Scale bars : 20 μm .

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Sections from control and Snai1 KO small intestine were stained for PCNA to detect proliferating cells (arrows depicting proliferating CBC stem cells at the base of crypts). Quantification of total PCNA‐positive cells per crypt and PCNA‐positive cells in the base of crypts both revealed a significant reduction in the number of proliferating cells after conditional depletion of Snai1 (n = 4, P = 0.049, 0.026). Scale bars: 20 μm.


expression of 2 CBCstem markers Olfm4 Lgr5 were examined by in situ hybridisation in control Snai1KO small intestine . Lgr5 Olfm4in situ hybridisation shows a loss of CBCstem marker expression after loss of Snai1 ( high magnification inserts depict a representative crypt demarcated by a dotted ) . Loss of Snai1 was confirmed in knockout tissue using immunohistochemistry . Scale bars : 20 μm .

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The expression of 2 CBCstem cell markers Olfm4 and Lgr5 were examined by in situ hybridisation in control and Snai1KO small intestine. Lgr5 and Olfm4in situ hybridisation staining shows a loss of CBCstem cell marker expression after loss of Snai1 (high magnification inserts depict a representative crypt demarcated by a dotted line). Loss of Snai1 was confirmed in knockout tissue using immunohistochemistry. Scale bars: 20 μm.


of sorted EGFP ‐ positive from Lgr5 ‐ EGFP ‐ IRES ‐ creERT2AhCreSnai1fl / fl Lgr5 ‐ EGFP ‐ IRES ‐ creERT2AhCreSnai1 + / + mice 5 days after βNF induction show an ˜ tenfold reduction in Lgr5GFP ‐ positive CBCstem .

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The analysis of sorted and EGFP‐positive cells from Lgr5‐EGFP‐IRES‐creERT2AhCreSnai1fl/fl and Lgr5‐EGFP‐IRES‐creERT2AhCreSnai1+/+mice 5 days after βNF induction show an ˜tenfold reduction in Lgr5GFP‐positive CBCstem cells.


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

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

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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 4 days of treatment compared to controls . Scale bars : 50 μm . of viable 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 .

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

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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.


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 . epithelium is re ‐ populated by wild ‐ type as shown on day 60 . Scale bars : 20 μm .

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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 was used to detect goblet in small intestine , no differences in numbers were observed in versus control mice ( n = 4 , NS ) .

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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 . of number of enteroendocrine per crypt - villus axes revealed a significant decrease in enteroendocrine numbers when Snai1 are elevated ( n = 4 , P = 0 . 003 ) .

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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 . of Paneth numbers indicated a significant decrease in Paneth in base of crypts in mice with elevated Snai1 ( n = 5 , P = 0 . 027 ) .

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

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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.


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

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

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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 localised at base of crypts ( depicted by arrows ) was observed . Bars represent mean ± SD . n = 3 mice per group , * P = 0 . 00023 . Two ‐ Student ' s t ‐ was used to assess significance . Scale bars : 10 μm .

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

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

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

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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 , which transiently express GFP ‐ SNAI1 , were used to detect direct binding of SNAI1 to promoters of known target genes CDH1 , IL8 a novel target SERINC3 revealed by microarray . An unrelated 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 . results are given as relative enrichment compared to input material are representative of three independent experiments .

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

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

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

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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 Cideb - / - mice were injected with AAV - GFP or AAV - Cideb , sacrificed for IB with indicated antibodies 20 days after injection .

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

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

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

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

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 lipogenic enzyme ( Fasn , Scd1 ) of WT Cideb - / - mice under chow diet HFLF diet .

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

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

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

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

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

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

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 SCAP in Golgi apparatus in Cideb - / - mice . IB of organelle fractions of indicated of IB data from 3 independent experiments . Data information : Data represent Mean ± ; NS : not significant , * : p < 0 . 05 , * * : p < 0 . 01 , * * * : p < 0 . 001 , by 2 - Student ' s t .

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

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

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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.


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

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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 ( PLIN2 , Insig , SCAP , SREBP - 1 or SREBP - 2 ) in 293T immuno - precipitated with an anti - Flag antibody , co - immunoprecipitated were detected by an anti - HA antibody following SDS - PAGE .

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

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

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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 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 SCAP or SCAP - Y298C in CHO - K1 in sterol - depleted medium . When indicated , 10 µg / ml 25 - HC were supplemented before were immuno - precipitated with an anti - Flag antibody , of co - immunoprecipitated were detected by IB following SDS - PAGE . dotted blue vertical was drawn to separate result between SCAP - WT SCAP - Y298C .

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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 , were treated with different doses of 25 - HC before subjected to IB with indicated antibodies following SDS - PAGE . of IB data from 3 independent experiments . Data represents Mean ± .

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

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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 transfected with Cideb GFP - tagged Sec12 , Sec23 , Sec16S , or Sec16L were subjected to immuno - precipitation with an anti - Cideb antibody , of co - immunoprecipitated were detected by an anti - GFP antibody .

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

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

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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 118 - 165 portion of Cideb . HA - tagged Sec12 was co - expressed with indicated Flag - tagged Cideb truncation mutants in 293T immuno - precipitated with an anti - Flag antibody . of co - immunoprecipitated 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 interaction with Sec12 . 293T transfected with HA - Sec12 indicated Flag - tagged Cideb constructs were subjected to immunoprecipitation with an anti - Flag antibody , of co - immunoprecipitated HA - Sec12 were detected with an anti - HA antibody .

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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 MBP - tagged Cideb or Cideb - K128A were purified from E . coli subjected to in vitro binding .

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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 , sacrificed 7 days after injection . Liver lysates were subjected to IB with indicated antibodies following SDS - PAGE of IB data from 3 independent experiments . Data represent Mean ± ; * * : p < 0 . 01 , * * * : p < 0 . 001 , by 2 - Student ' s t .

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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 indicated subjected to in vitro budding . Resulting vesicles donor microsomes were subject to IB with indicated antibodies following SDS - PAGE .

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

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

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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 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 indicated periods of time . Mitochondria were visualized via anti - Tom20 ( red ) . ( A ) PA accumulated on Parkin - positive mitochondria after 1 hour of 10uM CCCP treatment , with a more dramatic increase by 3hr 18 hr . Scale bar = 25 µM , zoom is 9x . ( B ) of imaging experiments shown in A B . Bars represent mean with from three biological independent experiments ; two - way ANOVA was performed for statistical ( * * * P < 0 . 0001 ) . ( C ) DAG ( diacylglycerol ) accumulated on Parkin - positive mitochondria after 5 . 5 hr of 10uM CCCP treatment . Arrowheads arrows point to DAGR at Golgi mitochondria , respectively . Scale bar = 25 µM , zoom is 9x .

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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 were transfected with Parkin - FLAG DAGR - YFP followed by CCCP treatment for indicated times . Mitochondrial fractions were purified analyzed by Western blots by indicated antibodies . Citrate synthetase α - tubulin were used as a mitochondrial marker , respectively . Note that PLD2 Parkin were elevated in mitochondrial fraction in response to CCCP treatment .

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

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

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

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

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(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 were transfected with control or a Lipin1 siRNA followed by expression plasmids of GFP - Parkin RFP - DAGR . Transfected 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 in single - channel blue in overlay ) . Arrows point to DAGR at Golgi under basal conditions . number ( E ) ( F , arbitrary unit ) of LC3 vesicles were quantified . n = 4 biological replicates . bars indicates mean ± . Asterisks indicate statistical significance by one - way ANOVA ( * P < 0 . 05 , * * P < 0 . 01 ) . Scale bar = 25 µM zoom is 5x . Note that both number of LC3 - vesicles in Lipin - 1 knockdown were much smaller than those in WT , these defects were corrected by DPG treatment .

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

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(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 were transfected with control or optineurin - NDP52 - siRNAs expression plasmids of FLAG - Parkin YFP - DAGR . These 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 zoom is 3x ) ( E ) Hela OPTN / NDP52 knockdown were transfected with a siRNA - resistant wildtype or ubiquitin - binding deficient E478G OPTN mutant , as indicated . of 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 . bars indicate mean ± . Bottom panel : Expression of OPTN , OPTN E478 NDP52 was determined by immunoblotting .

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

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(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 fractions obtained from control CCCP treated , were immunoblotted for EndoB1 OPTN , as indicated .

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


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

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

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

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(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 .

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


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

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


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

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(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 visualized by immuno ‐ electron microscopy . Black arrowhead indicates autophagosome . * , reference . Scale bar , 500 nm ( upper panel ) . WASH + / + or WASH − / − were isolated , LC3 conversion p62 level were analysed by immunoblotting ( lower panel ) . Bands were quantified analysed by Image J 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 online supplementary information page .

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(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 indicated times in presence or absence of 20 nM bafilomycin A1 ( BafA1 ) , harvested for immunoblotting . Ratios of LC3 ‐ II / β ‐ actin were calculated shown at right panel .

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(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 were visualized by with antibody against LC3 ( left panel ) , calculated as shown in right panel . Nuclei were stained with DAPI . Scale bar , 10 μm .

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

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(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 + / + 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 . * , reference . Fifty were quantified from three independent experiments ( right panel ) . Data were shown as means ± s . d . * P0 . 05 * * P0 . 01 . Experiments were repeated for three independent times with similar results . Source data for this figure is available on online supplementary information page .

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

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(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 stably expressing GFP ‐ LC3 were cultured with EBSS for 1 h stained with antibodies against EEA1 WASH .

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(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 stably expressing GFP ‐ Atg5 were transfected with Atg4B ( C74A ) mutant , followed by stimulation with EBSS for 1 h . were stained with anti ‐ WASH antibody ( red ) .

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(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 were treated with CM or 1 h in presence or absence of 20 nM BafA1 at 37 ° C for 1 h . WASH LAMP1 were visualized by with anti ‐ WASH anti ‐ LAMP1 antibodies .

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

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(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.