EMBO/soda-nel huggingface dataset:
An EZH2-dependent transcriptional complex promotes aberrant epithelial remodelling after injury (2021)
Figure 2

Figure 2. Polycomb mediates the fibrotic response in the co-culture system.

A. Hierarchical clustering of differentially regulated transcripts from RNA-seq between vehicle and TGFβ1-treated AECs in the co-culture (p-adj < 0.05, t-test with Benjamini-Hochberg Correction). Significantly regulated profibrotic genes (marked by asterisk) and histone/DNA methyltransferase encoded genes (marked in blue) are listed on the right side.

B. Volcano plot representing logarithmic ratio of differentially secreted proteins from proteomics analysis of co-culture medium upon apical TGFβ1 stimulation (p-adj < 0.05, t-test), with examples of profibrotic secreted proteins (red dots indicating significantly upregulated proteins, blue dots indicating significantly downregulated proteins, and grey dots indicating no significant change in protein expression levels).

C - E. Gene Ontology (GO) analysis of differentially expressed genes/proteins (p-adj < 0.05, hypergeometric test) that are enriched in (C) TGFβ1-treated, (D) vehicle treated AECs, and (E) differentially secreted proteins.

F. Gene set enrichment analysis (GSEA) shows enrichment of an IPF transcriptional and cellular phenotype in TGFβ1-injured AECs/MCs co-culture system (Kolmogorov-Smirnov test). Note, injured AECs displays an IPF transitional alveolar type 2 cells signature.

G. GSEA shows enrichment of genes defined as polycomb targets in injured AECs/MCs co-culture (Kolmogorov-Smirnov test with Benjamini-Hochberg Correction).

H. Representative H3K27me3 and EZH2 immunofluorescence images and box plots (minimum, first quartile, median, third quartile and maximum) showing decreased H3K27me3 levels but increased total EZH2 levels in TGFβ1-injured AECs in co-culture with MCs (n = 5 biological replicates with >50 cells per experiment, scale bars 50 µm, *p < 0.05, unpaired t-test).

See also Appendix Figure S2.
An EZH2-dependent transcriptional complex promotes aberrant epithelial remodelling after injury (2021)
Figure 3

Figure 3. TGFβ1-induced injury triggers an EZ-switch from EZH2-SUZ12 to EZH1-SUZ12 via EZH2-T311 phosphorylation in AECs.

A. Representative simple western analysis (Peggy Sue) of ph-EZH2 and quantification shows increased ph-EZH2 levels on T311 in AECs subjected to apical TGFβ1 for 72 h compared to vehicle treatment (mean + s.d., n = 5 biological replicates, ***p = 0.0008, unpaired t-test).

B. Representative simple western analysis (Peggy Sue) of SUZ12 immunoprecipitates shows co-precipitation of EZH1 and EZH2 in AECs. TGFβ1-induced injury leads to the EZ-switch from SUZ12-bound EZH2 to EZH1. Unspecific IgG binding was used as a negative control. A representative from 3 biological replicates is shown.

C. ChIP-qPCR shows increased ph-EZH2 occupancy at gene bodies of profibrotic genes in AECs subjected to TGFβ1 for 72 h. Note no changes in ph-EZH2 levels at non-target genes (mean + s.d., n = 3 biological replicates). Unspecific IgG was used as negative control. ChIP-qPCR for non-fibrotic genes is shown in Appendix Figure S3A.

D. ChIP-qPCR shows increased EZH1 occupancy at promoters of non-fibrotic genes in AECs subjected to apical TGFβ1 for 72 h (mean + s.d., n = 3 biological replicates). Unspecific IgG was used as negative control.

E. ChIP-qPCR shows no changes in H3K27me3 at promoters of non-fibrotic genes in AECs subjected to apical TGFβ1 for 72 h (mean + s.d., n = 3 biological replicates). Unspecific IgG was used as negative control.

F. Representative simple western analysis (Peggy Sue) and quantifications (right panels) for EZH2 and H3K27me levels from EZH2-deleted AECs (sgEZH2) which were reintroduced empty vector (EV), T311 wildtype (WT), a phosphorylated-deficient T311A, or a phosphomimetic T311D form of EZH2. Quantifications show mean + s.d. (n = 4 biological replicates, *p < 0.05, **p < 0.01, Kruskal-Wallis/Dunn's).

G. EZH2-deleted AECs (sgEZH2) reintroducing empty vector (EV), T311 wildtype (WT), a phosphorylated-deficient T311A, or a phosphomimetic T311D form of EZH2 were quantified for the expression of profibrotic genes. Vehicle and TGFβ1 treated AECs (sgNEG + vehicle / TGFβ1) were used as control. mRNA levels are normalised to HPRT1 expression. (mean + s.d., n = 4 biological replicates, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, Kruskal-Wallis/Dunn's).

See also Appendix Figure S3.
An EZH2-dependent transcriptional complex promotes aberrant epithelial remodelling after injury (2021)
Figure 4

Figure 4. Nuclear TAK1 mediates ph-EZH2 in injured AECs to control profibrotic response.

A. Kinase Enrichment Analysis showing enrichment of TAK1 (encoded by MAP3K7) in injured AECs (p-adj < 0.05, hypergeometric test with Benjamini-Hochberg Correction).

B. Nuclear fractionation followed by simple western analysis (Peggy Sue) of AECs exposed to 72 h of TGFβ1 in the co-culture system shows an increase in phosphorylated TAK1 and a parallel increase in ph-EZH2. Quantifications (lower panels) show mean + s.d., n = 3 biological replicates (ns = non-significant, ***p < 0.001, Kruskal-Wallis/Dunn's).

C. Simple western analysis (Peggy Sue) of EZH2 immunoprecipitates shows increased co-precipitation of ph-EZH2 (T311) and ph-TAK1 in injured AECs. Unspecific IgG was used as negative control. A representative from three experiments is shown.

D. Simple western analysis (Peggy Sue) shows a TAK1-dependent enrichment of ph-EZH2 levels in injured AECs. Note: TAK1 inhibitor (5-OZ) attenuates increased ph-EZH2 levels in injured AECs. Quantifications (right panels) show mean + s.d., n = 5 biological replicates (ns = non-significant, **p = 0.0026, ***p < 0.001, ANOVA/Tukey's).

E. ChIP-qPCR shows diminished POL2 occupancy on profibrotic genes in injured AECs subjected to 5-OZ (mean + s.d., n = 3 biological replicates). Unspecific IgG was used as negative control.

See also Appendix Figure S4.
An EZH2-dependent transcriptional complex promotes aberrant epithelial remodelling after injury (2021)
Figure 5

Figure 5. TGFβ1 mediates profibrotic response through a novel transcriptional complex of EZH2, POL2 and nuclear actin.

A. Nuclear fractionation followed by simple western analysis (Peggy Sue) shows an increase in nuclear actin in AECs exposed to TGFβ1 for 48 h. Quantification (right panel) shows mean + s.d., n = 3 biological replicates , ns = non-significant, *p = 0.014, ANOVA/Tukey's).

B. Simple western analysis (Peggy Sue) of EZH2 co-immunoprecipitates shows increased levels of EZH2-bound POL2, ph-EHZ2, and actin in injured AECs. Unspecific IgG binding was used as a negative control. A representative from 3 biological replicates is shown.

C, D. ChIP-qPCR shows increased occupancy of (C) POL2-S5p at promoters of profibrotic genes in AECs subjected to TGFβ1 for 24 h, whereas no enrichment of (D) POL2-S2p at the gene bodies of these genes was detected. Negative IgG control is shown in Appendix Figure S5B (mean + s.d., n = 3 biological replicates).

E, F. ChIP-qPCR shows increased occupancy of (E) POL2-S5p at promoters and (F) POL2-S2p at the gene bodies of profibrotic genes in AECs subjected to TGFβ1 for 48 h. Negative IgG control is shown in Appendix Figure S5C (mean + s.d., n = 3 biological replicates).

See also Appendix Figure S5.
An EZH2-dependent transcriptional complex promotes aberrant epithelial remodelling after injury (2021)
Figure 6

Figure 6. Actin dynamics regulate the profibrotic transcriptional response in injured AECs.

A. Representative immunofluorescence images of F-actin (phalloidin) and DAPI show that treatment with ROCK inhibitor Y27632 but not depletion of EZH2 can prevent TGFβ1-induced actomyosin remodelling in AECs (scale bars 200 µm).

B. Simple western analysis (Peggy Sue) of EZH2 immunoprecipitates shows abolition of TGFβ1-induced profibrotic transcriptional complex of EZH2/POL2/actin upon the convergent treatment of TGFβ1 and Y27632. Unspecific IgG binding was used as a negative control. Representative from 3 biological replicates is shown.

C, D. Simple western analysis (Peggy Sue) of nuclear fractionation (C) shows an increase in nuclear actin, ph-EZH2 and PO2-S2p levels in injured AECs. RNAi-mediated depletion of IPO9 (siIPO9) prevents injury-induced nuclear actin and POL2-S2p. Quantification (D) shows mean + s.d. (n = 3 biological replicates, *p < 0.05, **p < 0.01, ***p < 0.001, ANOVA/Tukey's).

E. qPCR analysis of profibrotic genes in MCs co-culture with AECs shows that depletion of IPO9 in TGFβ1-injured AECs blocks the fibrotic crosstalk with MCs. Data show mRNA levels of profibrotic genes normalised to S26 (mean + s.d., n = 3 biological replicates with 5 MCs donors, *p < 0.05, ***p < 0.001, ANOVA/Tukey's).

See also Appendix Figure S6.
An EZH2-dependent transcriptional complex promotes aberrant epithelial remodelling after injury (2021)
Figure 7

Figure 7. Non-canonical EZH2 regulates metaplastic differentiation of alveolar epithelium.

A. Simple western analysis (Peggy Sue) and quantifications (right panels) of mouse lung epithelial cells shows increased ph-EZH2 (T311), ph-TAK1, myosin activity (ph-MLC2) and POL2-K7m levels in AAV-mediated TGFβ1 over-expression. Note, increased ph-EZH2 and POL2-K7m levels are attenuated by the EZH2 inhibitor GSK126, whereas ph-TAK1 and ph-MLC2 levels cannot be rescued by GSK126. Quantifications (right panels) show violin plots, *p < 0.05, **p < 0.01, ***p < 0.001, ns = non-significant, Kruskal-Wallis/Dunn's.

B. Representative of 3D computed tomography (CT) reconstruction of the lung from control, AAV-TGFβ1 and GSK126-treated AAV-TGFβ1 mice (green: lung tissue, red: airways, and region-of-interest (ROI): blue). Insets show µCT slices in the middle of the lung from respective mice. Note, GSK126 attenuates TGFβ1-induced lung injury. Quantification (right panel) shows mean intensity of ROIs from the whole lung (violin plots, *p = 0.0385, **p = 0.0012, ANOVA/Holm-Sidak's).

C. qPCR analysis of differentiation genes in epithelial cells reveals that EZH2 is required for the effect of TGFβ1 on metaplastic differentiation gene expression. Data show mRNA levels of profibrotic genes normalised to S26 (violin plots, *p < 0.05, **p < 0.01, ***p < 0.001, ANOVA/Holm-Sidak's).

D. Immunofluorescence analysis of KRT5 as a marker for alveolar metaplastic basal cells and ph-EZH2 (scale bars 100 µm), pro-SFTPC as a marker for alveolar type 2 epithelial cells (scale bars 50 µm) and quantifications (right panels) show percentage of KRT5+ pods area per 10X field and percentage of pro-SFTPC+ cells per 20X field (*p < 0.05, unpaired t-test).

Data information: All violin plots display minimum, first quartile, median, third quartile and maximum; n = 5 control, 12 AAV-TGFβ1 and 13 GSK126-treated AAV-TGFβ1 mice.

See also Appendix Figure S7.
Cholesterol transfer via endoplasmic reticulum contacts mediates lysosome damage repair (2022)
Figure 2

Figure 2. Lysosome damage induces VAP-dependent ER-Lysosome contacts

A Representative electron micrographs of HeLa cells treated or not with 250 µM LLOMe for 5 min or 10 min before chemical fixation. Cells were incubated for 4 h following washing steps and overnight incubation with 15 nm gold nanoparticles conjugated to bovine serum albumin to visualize lysosomes (black spots inside lysosomes are internalized gold nanoparticles) before treatment with LLOMe and processing of samples for electron microscopy. Arrows indicate areas with increased lysosome-ER contacts.

B Quantification of electron micrographs showing an increase in membrane contact sites between ER and lysosomes following 250 µM LLOMe treatment for 5 or 10 minutes. Error bars denote +/- SD from n=3 independent experiments, >48 lysosomes were quantified per experiment for each condition. Statistical significance was determined using one-way ANOVA with Dunnett's multiple comparisons test, * p<0.05, ** p<0.01.

C Representative electron micrographs of VAP double knockout HeLa cells that were either left untreated or incubated with 250 µM LLOMe for 5 min or 10 min before chemical fixation. Cells were incubated with 15 nm gold nanoparticles conjugated to bovine serum albumin for 4 h to visualize lysosomes. After 4 h, gold nanoparticles were washed off and cells were incubated overnight before treatment with LLOMe and processing samples for electron microscopy (same as in A). Quantification of electron micrographs showing no increase in membrane contact sites between ER and lysosomes following 250 µM LLOMe treatment for 5 or 10 minutes in VAP double knockout cells. Error bars denote +/- SD from n=3 independent experiments, >40 lysosomes were quantified per experiment for each condition. Statistical significance was determined using one-way ANOVA with Tukey's multiple comparisons test, * p<0.05, ns= not statistically significant.

D Representative movie stills of a live-cell imaging experiment illustrating the recruitment of transiently expressed eGFP-ORP1L to lysosomes following 250 µM LLOMe treatment. Cells were pre-treated for 30 min with 75 nM Lysotracker Deep Red to monitor lysosome damage (judged by the decreased number of Lysotracker spots after LLOMe treatment). The graph represents quantification of eGFP-ORP1L foci per cell after LLOMe treatment. Error bars denote +/- SEM from n=3 independent live-cell imaging experiments, >25 cells were analyzed per experiment.
Cholesterol transfer via endoplasmic reticulum contacts mediates lysosome damage repair (2022)
Figure 3

Figure 3. ORP1L is recruited by PI4K2A-dependent accumulation of PtdIns4P on damaged lysosomes

A Movie stills from a live-cell imaging experiment showing rapid 2xSidM-eGFP recruitment to damaged lysosomes upon 250 µM LLOMe treatment (indicated in green). Lysosomes were visualized using transiently overexpressed LAMP1-mCherry (indicated in red). Yellow spots show colocalized PtdIns4P probe (2xSidM-eGFP) and LAMP1-mCherry after 3 and 10 min of LLOMe treatment. The graph shows the quantification of 2xSidM-eGFP foci per cell after LLOMe treatment. Error bars denote +/- SEM from n=3 independent live-cell imaging experiments, >20 cells were analyzed per experiment.

B HeLa cells were transfected with control siRNA or siRNA targeting PI4K2A or PI4K2B for 48 h (KD efficiency is shown in Fig EV5C), and transiently transfected with the 2xSidM-eGFP probe to monitor PtdIns4P recruitment after LLOMe-treatment by live-cell imaging. Representative movie stills show the dynamics of 2xSidM-eGFP recruitment in control, PI4K2A and PI4K2B depleted cells. The graph represents the quantification of 2xSidM-eGFP foci per cell after LLOMe-treatment. Error bars denote +/- SEM from n=3 independent live-cell imaging experiments, >25 cells were analyzed per experiment for each condition.

C CRISPR-Cas9 mediated knockout of PI4K2A abolishes recruitment of the 2xSidM-eGFP probe (PtdIns4P) to damaged lysosomes. Two different clones (clone 2 and clone 3) were tested. The graph represents the quantification of 2xSidM-eGFP foci per cell after LLOMe-treatment. Error bars denote +/- SD from n=2 independent live-cell imaging experiments, >20 cells were analyzed per experiment for each condition.

D Representative movie stills from live fluorescence microscopy showing reduced eGFP-ORP1L recruitment in PI4K2A knockout cells compared to wild type HeLa cells upon treatment with 250 µM LLOMe. The graph represents the quantification of eGFP-ORP1L foci per cell after LLOMe treatment. Error bars denote +/- SEM from n=4 independent live-cell imaging experiments, >80 cells were analyzed per experiment for each condition.
Cholesterol transfer via endoplasmic reticulum contacts mediates lysosome damage repair (2022)
Figure 4

Figure 4. PI4K2A mediates lysosome repair

Representative movie stills from live-cell imaging experiments monitoring Lysotracker recovery after induced lysosomal damage using 250 µM LLOMe. Cells were pre-treated with 75 nM Lysotracker Deep Red for 30 min before addition of LLOMe. The graph represents the quantification of bright Lysotracker positive foci per cell. While the decrease in the number of Lysotracker spots is quickly recovering in HeLa cells, there is a severe impairment in lysosomal repair in HeLa-PI4K2A-KO cells. Error bars denote +/-SEM from n=3 independent experiments, >217 cells were analyzed per experiment for each condition.
Cholesterol transfer via endoplasmic reticulum contacts mediates lysosome damage repair (2022)
Figure 5

Figure 5. The PI4K2A-mediated lysosome repair pathway is activated independently of ESCRTs

A HeLa cells were co-transfected with siRNAs targeting ESCRT proteins TSG101 and ALIX (siTSG101+siALIX) or with non-targeting siRNA control (siControl), and incubated for 24 h before transfection with the PtdIns4P binding probe 2xSidM-eGFP. 24 h post-transfection lysosomes were damaged with 250 µM LLOMe and PtdIns4P recruitment to damaged lysosomes was monitored using the 2xSidM-eGFP probe in live-cell imaging experiments. The graph shows quantification of 2xSidM-eGFP foci per cell in cells co-depleted of TSG101+ALIX and control (siControl) cells. Error bars denote +/- SD from n=2 independent live-cell imaging experiments, >27 cells were analyzed per experiment for each condition.

B Representative fluorescence micrographs of HeLa-PI4K2A knockout cells treated with 250 μM LLOMe or equal volume of DMSO (Pre) for 5 min, 15 min, 1 h before fixation and immunostained with Hoechst (blue), anti-CHMP4B (red), anti-GAL3 (green), and anti-ALIX (white).
Cholesterol transfer via endoplasmic reticulum contacts mediates lysosome damage repair (2022)
Figure 6

Figure 6. Cholesterol accumulation on damaged lysosomes depends on PI4K2A, VAPA/B and ORP1L

A Representative movie stills of a live-cell imaging experiment illustrate faster recruitment of the PtdIns4P probe 2xSidM-eGFP to damaged lysosomes when compared to cholesterol as indicated by the mCherry-D4 probe. The graph shows quantification of 2xSidM-eGFP and mCherry-D4 foci per cell. Error bars denote +/- SD from n=2 independent live-cell imaging experiments, >26 cells were analyzed per experiment for each condition.

B Graph showing quantification of mCherry-D4 and 2xSidM-eGFP foci per cell in parental HeLa cells (Ctrl) and PI4K2A knockout cells (PI4K2A-KO). Error bars correspond to +/- SEM from n=3 independent experiments, >30 cells per condition were analyzed.

C Quantification graph of 2xSidM-eGFP and mCherry-D4 foci per cell in HeLa and VAP double knockout cells. Error bars denote +/- SEM from n=3 independent experiments, >30 cells per condition were analyzed.

D HeLa cells expressing an eGFP-tagged ORP1L mutant incapable of binding to VAP (mFFAT) show no accumulation of the cholesterol reporter mCherry-D4 upon lysosomal damage induced with 250 µM LLOMe when compared to wildtype (wt) eGFP-ORP1L. The quantification graph shows D4-mCherry foci per cell. Error bars denote +/- SEM from n=4 independent live-cell imaging experiments, >50 cells were analyzed per experiment for each condition.
Cholesterol transfer via endoplasmic reticulum contacts mediates lysosome damage repair (2022)
Figure 7

Figure 7. Cholesterol accumulation protects lysosomes from damage

A Representative movie stills from live-cell imaging experiments indicate recruitment of CHMP4B-eGFP to damaged lysosomes in control and U18666A treated cells. HeLa cells stably expressing CHMP4B-eGFP were treated with 2 µM U18666A for 17 h before treatment with 250 µM LLOMe. The graph shows quantification of CHMP4B-eGFP foci per cell. Error bars denote +/- SEM from n=4, >30 cells per experiment for each condition.

B Representative immunofluorescence images illustrate Galectin-3 recruitment kinetics on the damaged lysosomes upon treatment with 2 µM U18666A for 17 h. HeLa cells were treated or not (Control) with U18666A and +/- LLOMe for the indicated time points, fixed and processed for immunofluorescence microscopy. Endogenous levels of Galectin-3 were visualized.

C Number of Galectin-3 positive dots per cell was automatically quantified from the dataset described in B. Error bars denote +/- SEM from n=3 independent experiments. >225 cells were analyzed per condition, distribution visualized as violin plots. One-way ANOVA, Tukey's multiple comparisons test, ** p<0.01, *** p<0.001, ns=not statistically significant.

D Representative movie stills from live-cell imaging experiments indicate recruitment of the PtdIns4P probe 2xSidM-eGFP to damaged lysosomes in control but not in the U18666A treated cells. HeLa cells were treated with 2 µM U18666A for 17 h before treatment with 250 µM LLOMe. The graph shows quantification of 2xSidM-eGFP foci per cell. Error bars denote +/- SEM from n=3 independent experiments, >25 cells per experiment for each condition.
Cholesterol transfer via endoplasmic reticulum contacts mediates lysosome damage repair (2022)
Figure 8

Figure 8. OSBP prevents hyperaccumulation of PtdIns4P on damaged lysosomes, promoting cell viability

A Representative movie stills of a live-cell imaging experiment show recruitment dynamics of transiently expressed eGFP-OSBP in HeLa cells treated with 250 µM LLOMe. The graph shows quantifications of OSBP foci per cell. Error bars denote +/- SEM from n=5 independent live-cell imaging experiments, >22 cells per experiment.

B Representative movie montage from a live-cell fluorescence imaging experiment indicating accumulation of the 2xSidM-eGFP probe and subsequent cell death in cells depleted of OSBP compared to Control following treatment with 250 µM LLOMe.

C Kaplan-Meier plot represents decrease in cell viability in siRNA knock down of OSBP (HeLa siOSBP) as compared to control cells (HeLa siControl). The rapid cell death observed in HeLa cells depleted of OSBP was ameliorated with PtdIns4P depletion (HeLa-PI4K2A-KO siOSBP and HeLa-PI4K2A-KO siControl). The graph represents data from n=3 independent live-cell imaging experiments, >100 cells per experiment for each condition.

D Knock down efficiency of siRNA depletion of OSBP in HeLa cells as detected by Western blot. β-actin used as a loading control.
USP7 and USP47 deubiquitinases regulate the NLRP3 inflammasome activation. (2018)
Figure 1

Figure 1. USP7 and USP47 inhibition with P22077 blocks human NLRP3 inflammasome activation.

IL-1β ELISA of supernatants from LPS-primed (1 μg/mL, 4hrs.) MDMs pre-incubated with 0.1% DMSO or P22077 (2.5 µM) 15 mins. before treatment with either nigericin (10 μM, 45 mins.) or CPPD crystals (250 μg/mL, 2 hrs.). Bars represent the mean, ± S.D. n = 13 and 11 independent blood donors for nigericin and CPPD, respectively. *** = P< 0.001 using a one-way ANOVA.

IL-18 ELISA of supernatants from MDMs treated as in A. Bars represent the mean, ± S.D. n = 11 independent blood donors. *** = P< 0.001, ** = P<0.01 and * = P<0.05 using a one-way ANOVA.

LDH-release from MDMs treated as in A. Bars represent the mean percentage of LDH release relative to the total cells lysed, ± S.D. n = 10 and 11 independent blood donors for nigericin and CPPD, respectively. *** P< 0.001; ** P< 0.01 using a one-way ANOVA. n.s. = not significant.

Western blots of supernatants and cell lysates from MDMs treated as in A. Bands in the figure represent: Pro-IL-1β (31 kDa); mature IL-1β (mIL-1β, 17 kDa); pro-IL-18 (24 kDa), mature IL-18 (mIL-18, 18 kDa), pro-caspase-1 (pro-Casp-1; 45 kDa) and mature caspase-1 (mCasp-1; 20 kDa). β-actin is shown as a loading control. Blots are representative of at least 3 independent blood donors.