EMBO/soda-nel huggingface dataset:
WASH inhibits autophagy through suppression of Beclin 1 ubiquitination (2013)
Figure 2

WASH inhibits autophagy. (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. (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.
WASH inhibits autophagy through suppression of Beclin 1 ubiquitination (2013)
Figure 3

WASH is localized in autophagosomes. (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. (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 EBSS 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. (F) Immuno‐electron microscopy analysis of HeLa 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.
WASH inhibits autophagy through suppression of Beclin 1 ubiquitination (2013)
Figure 4

WASH inhibits Vps34 activity. (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 1 h 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. (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. (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.
WASH inhibits autophagy through suppression of Beclin 1 ubiquitination (2013)
Figure 5

WASH associates with Beclin 1. (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. (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. (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. (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.
WASH inhibits autophagy through suppression of Beclin 1 ubiquitination (2013)
Figure 6

Lysine 437 ubiquitination of Beclin 1 is required for autophagy induction. (A) Endogenous Beclin 1 is ubiquitinated through K63 linkage during the process of autophagy. HeLa 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 immunoprecipitated 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. (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. (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‐WIPI1 were rescued with WT‐ or K437R‐Beclin 1, followed by stimulation with EBSS 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. (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.
Non-redundant functions of H2A.Z.1 and H2A.Z.2 in chromosome segregation and cell cycle progression (2021)
Figure 1

Figure 1. H2A.Z.2.1 knockdown leads to genome instability.

A) Representative images of HeLa cells treated with Control, H2A.Z.1 or H2A.Z.2 siRNA for 72 h, fixed, and stained with DAPI. White arrows point at micronuclei. Scale bar: 10 μm.

B) Quantification of the percentage of cells with micronuclei from experiment in (A). Three biological replicates were analysed for each condition (Control si N=1271; H2A.Z.1 si N=1091; H2A.Z.2#1 si N=1188; H2A.Z.2#2 si N=1193). The error bars represent the SD. Data sets were statistically analysed using Chi-square test. ***=p<0.001.

C) Quantification of the percentage of cells with micronuclei from HeLa cells co-transfected with H2A.Z.2 siRNA and GFP:H2A.Z.2.1WT with either single or double mutations. Error bars represent the SD of three biological replicates (Control si N=1316; H2A.Z.2 si N=1877; H2A.Z.2 si + wt N=1973; H2A.Z.2 si + A14T N=618; H2A.Z.2 si + S38T N=558; H2A.Z.2 si + A127V N=649; H2A.Z.2 si + A14T/S38T N=1090; H2A.Z.2 si + A14T/A127V N=1038; H2A.Z.2 si + S38T/A127V N=1150). Data sets were statistically analysed using Chi-square test. ***=p<0.001; black stars refer to the comparison with the H2A.Z.2si data and blue stars refer to the comparison with the H2A.Z.2si + wt data; no stars = non significant.

D) Schemes of the GFP constructs used for the rescue experiments in (E) and (F). Green boxes represent the GFP, brown boxes represent the H2A.Z.2.1 isoform. Solid fill represents the WT construct whereas striped box represents the KR mutant form where the residues indicate the mutations performed. Dots represent the possible post-translational modifications in the mutated amino acids: acetylation (ac, dark blue), methylation (me, pink) and ubiquitination (ub, light blue).

E) Representative images of prometaphase chromosomes from HeLa cells co-transfected with H2A.Z.2 siRNA and each of the constructs in D (green). Scale bar: 5μm.

F) Quantification of the percentage of cells with micronuclei from experiment (E). The error bars represent the SD of three biological replicates (Control si N=887; H2A.Z.2 si N=1005; H2A.Z.2 si + wt N=760; H2A.Z.2 si + KR N=479). Data sets were statistically analysed using Chi-square test. ***=p<0.001; ns= not significant. Blue stars refer to the comparison with the H2A.Z.2 si + wt data
Non-redundant functions of H2A.Z.1 and H2A.Z.2 in chromosome segregation and cell cycle progression (2021)
Figure 2

Figure 2. H2A.Z.2 regulates chromosome segregation.

A) Quantification of micronuclei containing 0, 1 or 2 CENP-A signals in HeLa YFP:CENP-A cells transfected with control and H2A.Z.2 siRNA for 72 h. 160 micronuclei were analysed. Error bars indicate SD of three biological replicates. The image represents an example of a micronucleus containing a single CENP-A signal. The insets are enlargements of the MN. Scale bar 5 μm.

B) Representative images of metaphases from YFP:CENP-A (green) HeLa cells treated with Control (top) or H2A.Z.2 (bottom) siRNA. The white arrowheads indicate the mis-aligned chromosomes. Scale bar: 10 μm.

C) Representative images of metaphase spreads from Control (top) or H2A.Z.2 (bottom) siRNA-transfected HeLa cells after FISH with a Chr17 centromeric probe (green). The arrowheads indicate the separated sister chromatids. Scale bar: 10 μm.

D) Representative images of HeLa cells transfected with Control (top) or H2A.Z.2 (bottom) siRNA, fixed, and stained for Sgo1 (green). Scale bar: 10 μm.

E) Quantification of Sgo1 localisation in pro-metaphase cells from the experiment in (D). Error bar represents SD of two biological replicates. 35 pro-metaphase cells were analysed.

F) Representative images of HeLa cells treated as in (D) and stained for Aurora B (green) and alpha tubulin (grey). Scale bar: 10 μm.

G) Mitotic index of HeLa cells transfected with Control (grey), H2A.Z.1 (blue) or H2A.Z.2 (red) siRNA and treated with nocodazole for 0, 2, 4 or 6 h. At least 800 cells were analysed for each category. Error bar represents SD of three biological replicates. **=p<0.01; ***=p<0.001 ns=not significant (Chi square test).

H) Left panels: Representative images of HeLa cells treated as in (D) and stained for HP1α (green). Scale bar: 10 μm. Right panels: Representative images of HeLa cells treated as in (D) and stained for h3T3ph (green). Scale bar 5 μm

I) Violin plot of centromeric H3T3ph intensity of pro-metaphase/metaphase cells from the experiment in (H). The median is shown as bar. Data sets were statistically analysed using the Wilcoxon rank test in R. **=p<0.01.

J) Representative images of HeLa YFP:CENP-A (green) mitotic cells after Control (top) or H2A.Z.2 (bottom) siRNA treatment. Scale bar: 5 μm.

K) Violin plot of centromeric CENP-A intensity of pro-metaphase/metaphase cells from the experiments in J. (Control si N=197 H2A.Z.2 si N=260). The bar represents the median. Data sets were statistically analysed using the Wilcoxon rank test in R. **=p<0.01.

L) Representative images of HeLa mitotic cells stained for CENP-C after Control (top) or H2A.Z.2 (bottom) siRNA treatment. Scale bar: 5 μm.

M) Violin plot of centromeric CENP-C intensity of pro-metaphase/metaphase cells from the experiments in L. (Control si N=1083 H2A.Z.2 si N=686, from 3 biological replicas). The bars represent the median. Data sets were statistically analysed using the Wilcoxon rank test in R. ***=p<0.0001.
Non-redundant functions of H2A.Z.1 and H2A.Z.2 in chromosome segregation and cell cycle progression (2021)
Figure 3

Figure 3. H2A.Z.1 regulates the expression of cell cycle genes.

A) Gene expression of three biological replicates of HeLa cells transfected with Control, H2A.Z.1 or H2A.Z.2 siRNA was analysed by RNA sequencing. Venn diagram shows the number of significant (p<0.01) differentially expressed genes and the overlap between each set of genes.

B) Pie charts displaying the percentages of H2A.Z peaks at or nearby transcription start sites (TSS) (dark blue), within the gene body (grey) or elsewhere in the whole genome (purple) for the differentially expressed genes following H2A.Z.1 or H2A.Z.2 depletion. A 2-sample test for equality of proportions was used for the statistical analyses. *=p<0.05; ns=not significant.

C) Downregulated genes following H2A.Z.1 depletion were analysed by STRING. The image shows the cluster of genes with gene onthology (GO) terms related to cell cycle. PPI=Protein-protein interaction.

D) IGV analyses of H2A.Z localisation (from ENCODE) on Aurora B (green) and c-MYC (blue) genes showing H2A.Z enrichment at the TSS and at the upstream region respectively (bottom panels). IGV analyses of ATAC seq peaks for Control si and H2A.Z.1 si for Aurora B and MYC genes. The number in red represents the log2Ratio H2AZ.1 si/Control si.

E) Plot representing the average value for all the target regions around the TSS obtained by ATAC seq after Control, H2A.Z.1 or H2A.Z.2 siRNAi.

F) Plot representing the number of differential chromatin accessibility regions in pairwise comparison. Green (up) represents regions with increased accessibility and red (down) with decreased accessibility.

G) Distribution of the position within the nucleus of the centromere of Chr17 from the experiment in (H) calculated as the ratio d2/d1. The ratio between d2 and d1 gives the position of the centromere relative to the center of the nucleus. The graph represents the percentages of centromeres with distances following within the 5 binning categories. At least 500 nuclei were analysed per condition. Data sets were statistically analysed using Chi-square test for the distribution of signals among the 5 categories. ***=p<0.001.

H) Representative image of a HeLa nucleus after FISH with Chr17 centromeric probe (green). The distance of the FISH signals from the nucleus periphery was calculated as follows: the distance from the centre of the nucleus to the periphery (d1) and the distance from the centre of the nucleus to the FISH signal (d2) were measured. (scale bar 5 μm).
Non-redundant functions of H2A.Z.1 and H2A.Z.2 in chromosome segregation and cell cycle progression (2021)
Figure 4

Figure 4. H2A.Z.1 depletion causes G1 arrest and cell senescence.

A) Flow cytometry analysis profiles of control (top) and H2A.Z.1 (bottom) siRNA-treated HeLa cells. Percentages represent the mean of two biological replicates. Data sets were statistically analysed using Chi-square test. ***=p<0.001.

B) CDKN1A (top) and CDKN1B (bottom) average expression values obtained by RNA sequencing of three biological replicates after Control, H2A.Z.1 (Z1) or H2A.Z.2 (Z2) siRNA treatment. Error bars show the standard deviation (SD). ***=p<0.001 (Student's t Test).

C) GSEA analyses of the upregulated (light blue upwards arrow) and downregulated (light green downwards arrow) genes upon H2A.Z.1 siRNA indicating the number of genes for category and the relative p values.

D) Representative images of HeLa cells transfected with control, H2A.Z.1 or H2A.Z.2 siRNA and stained for senescence-associated β-galactosidase (blue) Sclae bar 200μm. At the bottom-left, quantification of the percentage of β-galactosidase-positive cells. At least 500 cells were counted for each experiment. Error bars show the standard deviation (SD) from two biological replicates. **=p<0.01; ***=p<0.001 (Student's t Test).

E) Representative images of Hela cell stained for Ki67. The arrows indicate the different staining patterns quantified: strong signal (grey arrow), weak signal (light brown arrow) and no signal (dark brown arrow (scale bar 10μm).

F) Quantification of the experiment in (E). Error bars indicate the standard deviation from 3 biological replicates Data sets were statistically analysed using a Chi square test. ***=p<0.001.

G) Representative images of HeLa cells transfected with control (top) or H2A.Z.1 (bottom) siRNA and stained for Lamin A/C (green). Scale bar = 10μm.

H) Violin plots of the nuclear circularity index of HeLa cells transfected with control (grey), H2A.Z.1 (blue) or H2A.Z.2 (red) siRNA. The nuclear circularity was analysed using the NIS Elements AR analysis software (NIKON). At least 140 nuclei from three biological replicates were analysed for each condition. Mean and SD are shown. Data sets were statistically analysed using the Wilcoxon rank test in R. ***=p<0.001.

I) Violin plots of the nuclear circularity index of HeLa cells transfected with Control (grey) or H2A.Z.1 (blue) siRNA either alone or in combination with siRNA-resistant GFP:H2A.Z.1WT (WT) or GFP:H2A.Z.1KR (KR) plasmids. The nuclear circularity was analysed using the NIS Elements AR analysis software (NIKON). At least 140 nuclei from three biological replicates were analysed for each condition. Mean and SD are shown. Data sets were statistically analysed using the Wilcoxon rank test in R. ***=p<0.001.
Loss of TMEM106B potentiates lysosomal and FTD-like pathology in progranulin deficient mice (2020)
Figure 1

Figure 1. Early motor deficits in Grn-/-/Tmem106b-/- mice

Schema of the mouse wild type and mutant Tmem106b locus. The tm2a allele carries a LacZ-Neo selection cassette in intron 3 and a loxP-flanked exon 4. Upon Cre recombinase expression in the germline, exon 4 is deleted and Tmem106b expression abolished (tm2b allele).

Western blot analysis of total brain lysates of wild type, Tmem106b-/-, Grn-/- and Grn-/-/Tmem106b-/- mice using antibody 6F2 to detect TMEM106B. (4.5-month-old mice with the given genotype; n = 3 biological replicates per genotype).

Hind limb clasping test wild type, Tmem106b-/-, Grn-/-, Grn+/-/Tmem106b-/- and Grn-/-/Tmem106b-/- mice. Representative pictures are shown of 3 mice analyzed for each genotype at 3 months of age.

Rotarod performance of wild type, Tmem106b-/-, Grn-/-, Grn+/- /Tmem106b-/- and double knockout animals at 3 to 4 months of age. Number of animals used for analysis, 10 wild type, 12 Tmem106b-/-, 5 Grn-/-, 7 Grn+/-/Tmem106b-/-, 3 Grn-/-/Tmem106b-/- mice.

Representative picture of a 4.5-month-old double knockout (Grn-/-/Tmem106b-/- ) mouse after manual flip.

Data information: For statistical analysis (D), one-way ANOVA with Tukey's post hoc test was used to compare the mean ± SD of the individual genotypes. Significance is indicated; *p < 0.05; **p < 0.01; and ****p < 0.0001. Source data including exact p‐values are available online for this figure.