Journal: Science Advances

Article Title: EZH2 directly methylates PARP1 and regulates its activity in cancer

doi: 10.1126/sciadv.adl2804

Figure Lengend Snippet: ( A to C ) C4-2 cells (A), DU145 cells (B), or CRPC PDX LuCaP 35CR tissues (C) were lysed and collected for IP with anti-EZH2, PARP1, or immunoglobulin G (IgG), followed by IB analysis with indicated antibodies. ( D ) Purified proteins of GST-tagged EZH2 and His-tagged PARP1 were subjected to GST pull-down, followed by IB analysis. ( E ) Domain architecture of the EZH2 protein and its truncated mutants generated by IBS 2.0 . The homology domain 1 (H1) contains WDB domain, while the homology domain 2 (H2) contains the first SANT domain. ( F ) Co-IP of Flag-tagged PARP1 with full-length or truncated mutants of Myc-tagged EZH2, followed by IB analysis. ( G ) Domain organization of the PARP1 protein and its truncations generated by IBS 2.0 . ND, N-terminal domain; MD, middle domain; CD, C-terminal domain. ( H ) Co-IP of Myc-tagged EZH2 with truncated mutants of GST-tagged PARP1, followed by IB analysis. ( I ) Top, C4-2 cells were infected with control or two independent shEZH2 lentiviruses for 72 hours and subjected to Western blotting (WB). Bottom, quantification of relative PAR abundance normalized by H3 protein intensity. ( J ) Top, C4-2 cells were treated with either dimethyl sulfoxide (DMSO) or indicated doses of EZH2 inhibitor (EZH2i) for 3 days. Co-IP was performed with anti-PARP1 antibody and IB with indicated antibodies. Bottom, quantification of relative PARP1 autoPARylation abundance normalized by PARP1 protein intensity in IP samples. ( K ) Top, C4-2 cells were treated with either DMSO or indicated doses of EZH2i for 3 days. Co-IP was performed with anti-H2B antibody and IB with indicated antibodies. Bottom, quantification of relative H2B PARylation abundance normalized by H2B protein intensity in IP samples. All error bars represent means ± SD from n = 3 biologically independent experiments. P values were determined by unpaired two-tailed t test. ** P < 0.01, *** P < 0.001.

Article Snippet: Following procedures previously described in , 1 μg of recombinant GST-tagged EZH2 protein was mixed with 1 μg of recombinant His-tagged PARP1 protein in 1 ml of NP-40 lysis buffer (Thermo Fisher Scientific) with protease and phosphatase inhibitor cocktails (Thermo Fisher Scientific).

Techniques: Purification, Generated, Co-Immunoprecipitation Assay, Infection, Control, Western Blot, Two Tailed Test

Journal: Science Advances

Article Title: EZH2 directly methylates PARP1 and regulates its activity in cancer

doi: 10.1126/sciadv.adl2804

Figure Lengend Snippet: ( A ) Co-IP with anti-methylated lysine (Methyl K) antibody was performed in C4-2 and DU145 cells, followed by IB with anti-PARP1 or anti-H3 antibodies. Rabbit IgG was used as a negative control. ( B ) HEK293T and C4-2 cells were treated with either DMSO or indicated doses of EPZ-6438 for 3 days before co-IP with anti-PARP1 antibody. Methylation was detected by IB with anti–Methyl K antibody. ( C ) ESI-MS/MS fragmentation spectra of unmodified and methylated K486- and K607-containing peptides from proteolytically digestion by chymotrypsin. ( D ) Relative methylation percentage of the indicated lysine sites in HEK293T cell with or without EZH2i treatment by MS data analysis. ( E ) HEK293T cells were transfected with Flag-tagged PARP1-WT or -K607A. Co-IP was performed with anti-Flag antibody followed by IB with anti–Methyl K antibody, or reversely IP with anti–Methyl K antibody and IB with anti-Flag antibody. ( F ) C4-2 cells stably expressing Flag-tagged PARP1-WT or -K607A were subjected to IP with anti-Flag antibody and followed by IB with anti-K607me1 antibody. ( G ) C4-2 cells were treated with either DMSO or EZH2i EPZ-6438 for 3 days. IP was performed with anti-PARP1 antibody and IB with anti-K607me1 antibody. ( H ) Rescue assay followed by Co-IP to determine PARP1 K607 methylation level in EZH2-deficient C4-2 cells overexpressed with either WT or H689A-mutant EZH2. ( I ) Cell lysates were collected from EZH2 +/+ , EZH2 −/− , EED +/+ , and EED −/− XEN cells and subjected to Co-IP assay followed by WB to detect PARP1 K607me1. ( J ) Co-IP with anti-EZH2 followed by IB with anti-PAR, and Co-IP with anti-PARP1 followed by IB with anti-K607me1 were performed in C4-2 cells treated with 5 nM BMN 673 for 48 hours.

Article Snippet: Following procedures previously described in , 1 μg of recombinant GST-tagged EZH2 protein was mixed with 1 μg of recombinant His-tagged PARP1 protein in 1 ml of NP-40 lysis buffer (Thermo Fisher Scientific) with protease and phosphatase inhibitor cocktails (Thermo Fisher Scientific).

Techniques: Co-Immunoprecipitation Assay, Methylation, Negative Control, Tandem Mass Spectroscopy, Transfection, Stable Transfection, Expressing, Rescue Assay, Mutagenesis

Journal: Science Advances

Article Title: EZH2 directly methylates PARP1 and regulates its activity in cancer

doi: 10.1126/sciadv.adl2804

Figure Lengend Snippet: ( A ) Seesaw model for combinational strategy of EZH2 and PARP1 inhibition. ( B and C ) Quantification of synergistic potency and efficiency of EPZ-6438 and BMN 673 in C4-2 (B) and PC3 cells (C) by MuSyc . Left, dose-response surface. Middle, dose-response curve for BMN 673. Right, dose-response curve for EPZ-6438. The parameter alpha quantifies how the effective dose of one drug is altered by the presence of the other. In the case of synergistic potency, alpha >1. The parameter beta is defined as the percent increase in a drug combination’s effect beyond the most efficacious single drug. In the case of synergistic efficacy, beta >0. ( D ) C4-2 cell viability in the presence of the indicated concentrations of PARPi (talazoparib) along with DMSO or EZH2i (EPZ-6438). ( E ) PARP1 KD C4-2 cells with stably expression of PARP1-WT, -K607A, or -K607R were subjected to colony formation assay in the presence of the indicated concentrations of PARPi (talazoparib). ( F to H ) LuCaP 35CR PDX tumor growth in nude severe combined immunodeficiency mice received vehicle, EPZ-6438, BMN 673, or both for 28 days. Tumor weights (F) were measured at the end point. Tumor volumes (G) and body weights (H) were monitored on the indicated days. ( I ) WB for on-target validation. Tumor tissues were lysed and blotted with indicated antibodies. Protein levels were quantified and normalized against H3. ( J ) Left, representative images of IHC staining for Ki-67 and cleaved-caspase 3 (CC3). Right, quantification of IHC images by ImageJ. Error bars, means ± SD, n = 5, **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05; ns, not significant, as determined by unpaired two-tailed t test for (F), (I), and (J) or two-way ANOVA for (G).

Article Snippet: Following procedures previously described in , 1 μg of recombinant GST-tagged EZH2 protein was mixed with 1 μg of recombinant His-tagged PARP1 protein in 1 ml of NP-40 lysis buffer (Thermo Fisher Scientific) with protease and phosphatase inhibitor cocktails (Thermo Fisher Scientific).

Techniques: Inhibition, Stable Transfection, Expressing, Colony Assay, Immunohistochemistry, Two Tailed Test

Journal: Science Advances

Article Title: EZH2 directly methylates PARP1 and regulates its activity in cancer

doi: 10.1126/sciadv.adl2804

Figure Lengend Snippet: EZH2 directly interacts with and methylates PARP1 at lysine 607. EZH2 inhibition or PARP1 K607 mutation increases PARP1 poly(ADP-ribose) polymerase activity, fine-tunes PARP1 functions in DDR, transcription, and tumor progression.

Article Snippet: Following procedures previously described in , 1 μg of recombinant GST-tagged EZH2 protein was mixed with 1 μg of recombinant His-tagged PARP1 protein in 1 ml of NP-40 lysis buffer (Thermo Fisher Scientific) with protease and phosphatase inhibitor cocktails (Thermo Fisher Scientific).

Techniques: Inhibition, Mutagenesis, Activity Assay

Journal: iScience

Article Title: EZH2 T367 phosphorylation activates p38 signaling through lysine methylation to promote breast cancer progression

doi: 10.1016/j.isci.2022.104827

Figure Lengend Snippet: EZH2 methylates p38α, and EZH2 phosphorylation at T367 is critical for p38α methylation and phosphorylation in TNBC (A) Co-immunoprecipitation (co-IP) and immunoblots of methylated p38α in a panel of breast cancer cells. EZH2 shRNA knockdown decreased methylated p38α. (B) Lysine N-methyltransferase (KMTase) activity assay in whole-cell lysates of MDA-MB-231 cells at the indicated conditions. KMTase activity was assessed using 0.1 mg/mL of human recombinant p38α and measuring S-adenosyl homocysteine (SAH) production detected by bioluminescence. shEZH2 and EPZ significantly reduced methylated p38α compared to control (lanes 1–3), which was rescued by WT-EZH2 (lane 4). EPZ reduced methylated p38α compared to WT-EZH2 (lanes 4–5). Bars depict mean ± SEM, ∗p ≤ 0.05. (C) KMTase activity assay in whole-cell lysates of T4 and Vari068 patient-derived TNBC cells control and treated with EPZ as in (B). Bars show mean ± SEM, ∗p ≤ 0.05. (D) IP and immunoblots of methylated p38α in MDA-MB-231 cells transduced with scrambled shRNA (control) or 3′ UTR EZH2-targeting shRNA (shEZH2) rescued with Myc-tagged WT-EZH2, T367A-EZH2, or vector (pBabe). (E) Immunoblot of cells in (D).

Article Snippet: Recombinant GST-EZH2 , BPS Bioscience , Cat #50279.

Techniques: Methylation, Immunoprecipitation, Co-Immunoprecipitation Assay, Western Blot, shRNA, Activity Assay, Recombinant, Derivative Assay, Transduction, Plasmid Preparation

Journal: iScience

Article Title: EZH2 T367 phosphorylation activates p38 signaling through lysine methylation to promote breast cancer progression

doi: 10.1016/j.isci.2022.104827

Figure Lengend Snippet: EZH2 methylates p38α protein at lysine 139 and lysine 165 leading to enhanced p38α protein stability (A) Schematic representation of p38 protein indicating the position of its functional domains and methylation sites (red dots). The amino acid positions of each domain are indicated below the structures. The table summarizes the unique sites of methylation identified by LC-MS-MS analyses of methylated proteins. Recombinant histone H3 and GST-p38α were incubated with or without recombinant PRC2 complex (EZH2/EED/SUZ12/RbAp48/AEBP2) and S-adenosyl methionine methyl donor in sodium phosphate-buffered HMTase buffer solution for one hour at 37°C. Samples were subsequently run on a gel and digested in-gel using trypsin or Arg-C and analyzed for monomethylation, dimethylation, and trimethylation by LC-MS/MS. The presence of methylation was confirmed by β- and γ-ion. (B) Pulse-chase analysis of MDA-MB-231 shVector and shEZH2 treated with 100 μg/mL of cycloheximide (CHX) at the indicated time points. Cell extracts were immunoblotted with anti-EZH2 and anti-p38α. α-Tubulin was used as the loading control. (C) Pulse-chase analysis for MDA-MB-231 treated with vehicle (control) or GSK-343 (1 μM) for 48 h and treated with 100 μg/mL of cycloheximide (CHX) at the indicated time points. Cell extracts were immunoblotted with anti-EZH2 and anti-p38α. α-Tubulin was used as the loading control. (D) HA-tagged p38α wild-type and p38α mutants K139A, K165A, and K139A/K165A were transduced into MDA-MB-231 cells and subjected to IP and WB using anti-p38α and anti-pan methyl-K antibody. Actin was used as the loading control. (E) CHX pulse-chase assay of cells in (D). (F) Ubiquitination assay. Indicated cells were treated with the proteasome inhibitor MG-132 (50 μM) for 5 h or vehicle. Whole-cell extracts were subsequently immunoprecipitated by anti-magnetics A beads followed by immunoblot using antibodies against ubiquitin and p38α. (G) Invasion assay of MDA-MB-231 cells transduced with HA-tagged p38α wild-type and p38α mutants K139A, K165A, and K139A/K165A. Scale bar, 10 μm. Bars show mean ± SEM, ∗p ≤ 0.05.

Article Snippet: Recombinant GST-EZH2 , BPS Bioscience , Cat #50279.

Techniques: Functional Assay, Methylation, Liquid Chromatography with Mass Spectroscopy, Recombinant, Incubation, Pulse Chase, Ubiquitin Assay, Immunoprecipitation, Western Blot, Invasion Assay, Transduction

Journal: iScience

Article Title: EZH2 T367 phosphorylation activates p38 signaling through lysine methylation to promote breast cancer progression

doi: 10.1016/j.isci.2022.104827

Figure Lengend Snippet: Combined pharmacological blockade of EZH2 and p38 enzymatic activities reduces neoplastic functions (A) Immunoblots of MDA-MB-231 cells treated with GSK-343 (3 μM for 48 h), SB202190 (p38i, 20 μM for 48 h), or the combination. (B) Cells in A were subjected to growth assays. (C) Synergistic effect of EZH2 inhibitor and p38 inhibitor in MDA-MB-231 cells incubated with various doses of GSK-343 and SB202190 for 4 d. A matrix for synergy score was calculated ( Ianevski et al., 2017 ). (D and E) Wound healing assay to quantify cell migration (D) and reconstituted Boyden basement membrane-invasive chamber assay of MDA-MB-231 cells treated as in (A). Representative chambers after crystal violet staining are shown above bars. Data for B-E are from at least three independent experiments carried out in at least triplicate. Data for B, (D and E) are presented as mean ± SEM. ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.005; ∗∗∗∗p ≤ 0.0001.

Article Snippet: Recombinant GST-EZH2 , BPS Bioscience , Cat #50279.

Techniques: Western Blot, Incubation, Wound Healing Assay, Migration, Boyden Chamber Assay, Staining

Journal: iScience

Article Title: EZH2 T367 phosphorylation activates p38 signaling through lysine methylation to promote breast cancer progression

doi: 10.1016/j.isci.2022.104827

Figure Lengend Snippet: Combined targeting of EZH2 and p38 enzymatic activities reduces primary breast cancer growth and metastasis (A) Primary tumor growth curves of NOD/SCID mice orthotopically implanted with MDA-MB-231 cells. When primary tumors reached 100 mm 3 , mice were treated intraperitoneally with EPZ-6438 (10 mg/kg/day), SB202190 (p38i, 1 mg/kg/day), combination, or control (4% DMSO-30% PEG 300-5% Tween 80), 5 days/week for 56 days (n = 10/group). Primary tumor growth as assessed by caliper measurements, shown as mean ± SEM. (B) Quantification of tumor volume at day 56 shown as mean ± SEM. (C) MDA-MB-231 cells were injected intracardially in nude mice (n = 10/group) and treated as in (A), for 3 weeks. Bars show the number of metastases per mouse in each group on day 21 after heart inoculation ±SEM. (D) Representative H&E-stained sections of lung metastases. Magnification 600x. Scale bar 50 μm. (E) Co-immunoprecipitation and immunoblots of methylated p38α in whole-cell lysates of primary MDA-MB-231 xenograft tumors derived from (A). (F) Immunoblots for the indicated proteins in lysates obtained from the MDA-MB-231 primary orthotopic xenografts in (A). For A-C, ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.005; ∗∗∗∗p ≤ 0.0001.

Article Snippet: Recombinant GST-EZH2 , BPS Bioscience , Cat #50279.

Techniques: Injection, Staining, Immunoprecipitation, Western Blot, Methylation, Derivative Assay

Journal: iScience

Article Title: EZH2 T367 phosphorylation activates p38 signaling through lysine methylation to promote breast cancer progression

doi: 10.1016/j.isci.2022.104827

Figure Lengend Snippet: Combined inhibition of EZH2 methyltransferase and p38 kinase activities reduce AKT signaling in vivo (A) RNA sequencing studies of mammary tumors treated with EPZ-6438 (10 mg/kg/day), SB202190 (p38i, 1 mg/kg/day), combination, or control (4% DMSO-30% PEG 300-5% Tween 80) and excised at day 56. The graph shows significantly deregulated pathways by the combination EPZ/p38i vs. control. (B) Immunoblot of primary xenografts treated as indicated in (A). Combined EPZ/p38i reduces p -AKT compared to single inhibitors. (C) Immunoblots for pAKT and total AKT in MDA-MB-231 cells transduced with HA-tagged WT-p38α, K139A-p38α, K165A-p38α, and K139A/K165A-p38α. P38α mutants display reduced pAKT levels compared to WT-p38α. (D) Immunoblots of MDA-MB-231 EZH2 KD rescued with Myc-tagged WT-EZH2, T367A-EZH2, or vector (pBabe) show that T367 phosphorylation is necessary to upregulate pAKT1 without changes in total AKT1. (E) Representative images of human primary invasive carcinomas. Case 1 shows an invasive high-grade ductal carcinoma with concordant high cyto-pEZH2-T367 and high pAKT, while case 2 shows an intermediate-grade invasive carcinoma with low expression of both proteins. Bars, 50 μm. (F) Schematic illustrating our working model of EZH2 function in breast cancer through H3K27me3-dependent and independent activities. P and Me represent phosphorylation and methylation, respectively.

Article Snippet: Recombinant GST-EZH2 , BPS Bioscience , Cat #50279.

Techniques: Inhibition, In Vivo, RNA Sequencing Assay, Western Blot, Transduction, Plasmid Preparation, Expressing, Methylation

Journal: iScience

Article Title: EZH2 T367 phosphorylation activates p38 signaling through lysine methylation to promote breast cancer progression

doi: 10.1016/j.isci.2022.104827

Figure Lengend Snippet:

Article Snippet: Recombinant GST-EZH2 , BPS Bioscience , Cat #50279.

Techniques: Plasmid Preparation, Derivative Assay, Recombinant, In Situ, Staining, RNA Sequencing Assay, ChIP-sequencing, shRNA, Software

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