Journal: EMBO Reports

Article Title: An EZH2‐dependent transcriptional complex promotes aberrant epithelial remodelling after injury

doi: 10.15252/embr.202152785

Figure Lengend Snippet: 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 Fig S2.

Article Snippet: In brief, recombinant human EZH2 protein (Novus Biologicals, H00002146‐P01) was incubated with recombinant TAK1 protein (Novus Biologicals, H00006885‐P01) in kinase buffer (Cell Signaling, 9802S), supplemented with 300 μM ATP (Cell Signaling, 9804) for 60 min at 30°C.

Techniques: RNA Sequencing, Co-Culture Assay, Expressing, Immunofluorescence

Journal: EMBO Reports

Article Title: An EZH2‐dependent transcriptional complex promotes aberrant epithelial remodelling after injury

doi: 10.15252/embr.202152785

Figure Lengend Snippet: 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 Fig 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 Fig S3. Source data are available online for this figure.

Article Snippet: In brief, recombinant human EZH2 protein (Novus Biologicals, H00002146‐P01) was incubated with recombinant TAK1 protein (Novus Biologicals, H00006885‐P01) in kinase buffer (Cell Signaling, 9802S), supplemented with 300 μM ATP (Cell Signaling, 9804) for 60 min at 30°C.

Techniques: Simple Western, Binding Assay, Negative Control, ChIP-qPCR, Plasmid Preparation, Expressing, Control

Journal: EMBO Reports

Article Title: An EZH2‐dependent transcriptional complex promotes aberrant epithelial remodelling after injury

doi: 10.15252/embr.202152785

Figure Lengend Snippet: 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 Fig S4. Source data are available online for this figure.

Article Snippet: In brief, recombinant human EZH2 protein (Novus Biologicals, H00002146‐P01) was incubated with recombinant TAK1 protein (Novus Biologicals, H00006885‐P01) in kinase buffer (Cell Signaling, 9802S), supplemented with 300 μM ATP (Cell Signaling, 9804) for 60 min at 30°C.

Techniques: Fractionation, Simple Western, Co-Culture Assay, Negative Control, ChIP-qPCR

Journal: EMBO Reports

Article Title: An EZH2‐dependent transcriptional complex promotes aberrant epithelial remodelling after injury

doi: 10.15252/embr.202152785

Figure Lengend Snippet: 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 Fig 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 Fig S5C (mean + s.d., n = 3 biological replicates). See also Appendix Fig S5. Source data are available online for this figure.

Article Snippet: In brief, recombinant human EZH2 protein (Novus Biologicals, H00002146‐P01) was incubated with recombinant TAK1 protein (Novus Biologicals, H00006885‐P01) in kinase buffer (Cell Signaling, 9802S), supplemented with 300 μM ATP (Cell Signaling, 9804) for 60 min at 30°C.

Techniques: Fractionation, Simple Western, Binding Assay, Negative Control, ChIP-qPCR, Control

Journal: EMBO Reports

Article Title: An EZH2‐dependent transcriptional complex promotes aberrant epithelial remodelling after injury

doi: 10.15252/embr.202152785

Figure Lengend Snippet: Representative simple western analysis (Peggy Sue) shows histone fraction (upper panel) and non‐histone fraction (lower panel) from TGFβ1‐injured AECs and control. These cells were further treated with an EZH2 inhibitor GSK126. Note the loss of ph‐EZH2 in GSK126‐treated AECs. Quantifications (right panels) shows mean + s.d. ( n = 5 biological replicates, *P < 0.05, * *P = 0.01, ns = non‐significant, Friedman/Dunn’s test for H3K27me3, ANOVA /Sidak´s test for ph‐EZH2). Representative simple western analysis (Peggy Sue) shows increased POL2‐K7 methylation (K7m) levels in injured AECs. This increase is blocked by GSK126. Quantification (right panel) shows mean + s.d. ( n = 5 biological replicates, *P < 0.05, ANOVA/Tukey’s). Simple western analysis (Peggy Sue) of EZH2 co‐immunoprecipitates shows increased levels of EZH2‐bound POL2‐K7m and decreased levels of EZH2‐bound SUZ12 in injured AECs. Unspecific IgG binding was used as a negative control. A representative from 3 biological replicates is shown. ELISA of profibrotic markers shows that inhibition of EZH2 activity by GSK126 attenuates the profibrotic effect of injured AECs on MCs ( n = 3 biological replicates from 5 MCs donors, mean + s.d., *P < 0.05, * *P < 0.01, ** *P < 0.001, ANOVA/Tukey´s). Source data are available online for this figure.

Article Snippet: In brief, recombinant human EZH2 protein (Novus Biologicals, H00002146‐P01) was incubated with recombinant TAK1 protein (Novus Biologicals, H00006885‐P01) in kinase buffer (Cell Signaling, 9802S), supplemented with 300 μM ATP (Cell Signaling, 9804) for 60 min at 30°C.

Techniques: Simple Western, Control, Methylation, Binding Assay, Negative Control, Enzyme-linked Immunosorbent Assay, Inhibition, Activity Assay

Journal: EMBO Reports

Article Title: An EZH2‐dependent transcriptional complex promotes aberrant epithelial remodelling after injury

doi: 10.15252/embr.202152785

Figure Lengend Snippet: 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 Fig S6. Source data are available online for this figure.

Article Snippet: In brief, recombinant human EZH2 protein (Novus Biologicals, H00002146‐P01) was incubated with recombinant TAK1 protein (Novus Biologicals, H00006885‐P01) in kinase buffer (Cell Signaling, 9802S), supplemented with 300 μM ATP (Cell Signaling, 9804) for 60 min at 30°C.

Techniques: Immunofluorescence, Simple Western, Binding Assay, Negative Control, Fractionation, Co-Culture Assay

Journal: EMBO Reports

Article Title: An EZH2‐dependent transcriptional complex promotes aberrant epithelial remodelling after injury

doi: 10.15252/embr.202152785

Figure Lengend Snippet: 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 overexpression. 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 Fig S7. Source data are available online for this figure.

Article Snippet: In brief, recombinant human EZH2 protein (Novus Biologicals, H00002146‐P01) was incubated with recombinant TAK1 protein (Novus Biologicals, H00006885‐P01) in kinase buffer (Cell Signaling, 9802S), supplemented with 300 μM ATP (Cell Signaling, 9804) for 60 min at 30°C.

Techniques: Simple Western, Activity Assay, Over Expression, Computed Tomography, Control, Gene Expression, Immunofluorescence, Marker

Journal: EMBO Reports

Article Title: An EZH2‐dependent transcriptional complex promotes aberrant epithelial remodelling after injury

doi: 10.15252/embr.202152785

Figure Lengend Snippet: TGFβ1‐injured epithelium activates TAK1 and actomyosin remodelling, which subsequently induces nuclear translocation of TAK1 and actin. Nuclear TAK1 mediates the phosphorylation of EZH2 on T311 and facilitates the release of EZH2 from PRC2. The liberation of EZH2 is accompanied by an EZ switch to EZH1‐PRC2, which is required to maintain H3K27me3 at TGFβ1 non‐target genes. Simultaneously, EZH2 establishes the fibrotic transcriptional complex with POL2 and nuclear actin to promote the metaplastic differentiation of AECs and triggers the fibrotic crosstalk with MCs. Perturbing this fibrotic complex blocks the fibrotic cascade, reinforces tissue repair and restores homeostasis.

Article Snippet: In brief, recombinant human EZH2 protein (Novus Biologicals, H00002146‐P01) was incubated with recombinant TAK1 protein (Novus Biologicals, H00006885‐P01) in kinase buffer (Cell Signaling, 9802S), supplemented with 300 μM ATP (Cell Signaling, 9804) for 60 min at 30°C.

Techniques: Translocation Assay, Phospho-proteomics

Journal: International journal of cancer

Article Title: Polycomb group proteins EZH2 and EED directly regulate androgen receptor in advanced prostate cancer

doi: 10.1002/ijc.32118

Figure Lengend Snippet: PRC2 protein EED and EZH2 interact with AR. (a) Immunoprecipitation of VCaP cell lysates with the indicated mouse monoclonal anti-EED antibody (05–1,320, Millipore), rabbit polyclonal anti-EED antibody (09–774, Millipore), control IgG and anti-AR antibody was followed by immunoblot analysis. Representative graph from at least three independent experiments is shown. (b) Immunoprecipitation of 22Rv1,C4–2, LNCaP and VCaP cell lysates with anti-EZH2, anti-AR antibody and control IgG was followed by immunoblot analysis. (c) HEK293T cells transfected with Halo-AR (full length), Halo-DBD, Halo-LBD, Halo-NTD plasmids and empty vector were lysed and subjected to pull-down assay using HaloLink resin (Promega), followed by immunoblot analysis. (d) Purified EZH2 and EED were respectively mixed with AR (full length) and pulled down with anti-AR antibody and protein A beads. RING1B served as a negative control. (e) Purified EED was mixed with AR N-terminal fragment and pulled down with anti-AR antibody and protein A beads. RING1B served as a negative control.

Article Snippet: For in vitro immunoprecipitation, AR-FL (346101–5,000 U, EMD Millipore), EZH2 (50,279, BPS Bioscience), EED (50,280, BPS Bioscience) and AR-NTD (ab82124, Abcam) were purchased from the vendor listed.

Techniques: Immunoprecipitation, Western Blot, Transfection, Plasmid Preparation, Pull Down Assay, Purification, Negative Control

Journal: International journal of cancer

Article Title: Polycomb group proteins EZH2 and EED directly regulate androgen receptor in advanced prostate cancer

doi: 10.1002/ijc.32118

Figure Lengend Snippet: EZH2 and EED knockdown decreases AR and downstream targets. (a) EZH2 was depleted by shRNA in C4–2 cells. After 48 hr, cells were lysed and blotted by EZH2, EED (rabbit polyclonal anti-EED antibody, 09–774, Millipore), AR, PSA and GAPDH. (b) EED was depleted by shRNA in C4–2 cells. After 48 hr, cells were lysed and blotted by EZH2, EED (rabbit polyclonal anti-EED antibody, 09–774, Millipore), AR, PSA and GAPDH. (c) LNCaP and VCaP cells were subjected to cotransfection of PSA or TMPRSS2 firefly luciferase reporter constructs and pRL-TK (Renilla luciferase). Lentivirus packaged with two distinct shRNAs of EZH2 or EED were added 24 hr after the cotransfection to knockdown EZH2 or EED. The luciferase activity was normalized using Renilla bioluminescence.

Article Snippet: For in vitro immunoprecipitation, AR-FL (346101–5,000 U, EMD Millipore), EZH2 (50,279, BPS Bioscience), EED (50,280, BPS Bioscience) and AR-NTD (ab82124, Abcam) were purchased from the vendor listed.

Techniques: shRNA, Cotransfection, Luciferase, Construct, Activity Assay

Journal: International journal of cancer

Article Title: Polycomb group proteins EZH2 and EED directly regulate androgen receptor in advanced prostate cancer

doi: 10.1002/ijc.32118

Figure Lengend Snippet: EZH2 knockdown and astemizole treatment demonstrate similar inhibition patterns of AR signaling blockage.(a) EZH2 knockdown and astemizole-treated samples cluster together based on log expression of 1,571 (top 10%) high variation genes. (b) Heat maps for the expression level of genes down- or up-regulated by EZH2 knockdown, GSK126 and astemizole treatment. (c) The number of overlapped differential genes in each paired group is significantly larger than the number of genes overlapped by chance. (d) 426 AR-induced genes were compared and the expression is similar between EZH2 knockdown and astemizole-treated samples. (e) Comparison of PSA gene track between groups. (f) Comparison of PSA gene track between groups. (g) GSEA shows that AR target genes are significantly enriched (Q value = 0.0429) in downregulated genes due to EZH2 knockdown. (h) GSEA shows that AR target genes are significantly enriched (Q value = 0.0413) in downregulated genes due to astemizole treatment.

Article Snippet: For in vitro immunoprecipitation, AR-FL (346101–5,000 U, EMD Millipore), EZH2 (50,279, BPS Bioscience), EED (50,280, BPS Bioscience) and AR-NTD (ab82124, Abcam) were purchased from the vendor listed.

Techniques: Inhibition, Expressing

Journal: International journal of cancer

Article Title: Polycomb group proteins EZH2 and EED directly regulate androgen receptor in advanced prostate cancer

doi: 10.1002/ijc.32118

Figure Lengend Snippet: Astemizole has potent therapeutic effects on prostate cancer. (a) Astemizole critically thwarts cell proliferation in C4–2 and other AR-positive prostate cancer cell lines. (b) The wound healing assay indicates that astemizole compromises the migration of C4–2 cells. (c) Astemizole decreases the invasive abilities of C4–2 cells compared to vehicle treatment. Cell count was analyzed and the difference was statistically significant. (d)C4–2 cells were treated with 2.5, 5 and 7.5 μM of astemizole. Cells were lysed 48 hr after treatment and blotted with anti-LC3-A/B antibody. The ratio of LC3-A/B-II/I to GAPDH was elevated as dose increased, which indicates that astemizole induces autophagy in prostate cancer cells. (e) Castration-resistant VCaP xenograft mouse models were generated. Castrated mice bearing CPRC xenografts received vehicle or astemizole treatment (50 mg kg−1) daily (5 days per week). Caliper measurements were taken every 4 days to determine tumor volume. Mean tumor volume SEM, *p < 0.05, **p < 0.01 vs. vehicle was marked. (f) Kaplan–Meier survival plot compares progression-free survival. (g) Upper panel: Proteins were blotted and quantitated to compare the protein levels of EZH2 and AR in astemizole-treated group (n = 8) compared to vehicle-treated group (n = 12). Lower panel: The expression of EZH2 and AR was decreased in response to astemizole treatment. (h) The proportion of the cells stained with EZH2/AR/PSA in astemizole-treated group (n = 6) were significantly lower than that in vehicle-treated group (n = 6).

Article Snippet: For in vitro immunoprecipitation, AR-FL (346101–5,000 U, EMD Millipore), EZH2 (50,279, BPS Bioscience), EED (50,280, BPS Bioscience) and AR-NTD (ab82124, Abcam) were purchased from the vendor listed.

Techniques: Wound Healing Assay, Migration, Cell Counting, Generated, Expressing, Staining