human recombinant ezh2 protein #tp302054  (OriGene)

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: bioRxiv

Article Title: A polycomb-independent role of EZH2 in TGFβ1-damaged epithelium triggers a fibrotic cascade with mesenchymal cells

doi: 10.1101/2020.07.29.225300

Figure Lengend Snippet: (a) EPCs were subjected to apical TGFβ1 stimulation for 72 h in the co-culture system. (b) Representative E-cadherin (E-cad) and DAPI immunofluorescence images of EPCs treated apically with TGFβ1 or vehicle control after 72 h in the co-culture system (scale bars 25μm). (c) ELISA analysis shows decreased E-cad levels in TGFβ1-treated EPCs after 72 h in the mono- and co-culture systems (n = 6 independent experiments, mean + s.d., ***p < 0.001, ANOVA/Tukey’s). (d) Representative COL-1, αSMA and DAPI staining of NHLFs from the mono- and co-culture systems after 72 h apical treatment with TGFβ1. Note the increased COL-1 levels, further enhanced by the addition of EPCs and an increase in αSMA levels only in the presence of EPCs (scale bars 200μm). (e) ELISA analysis shows increased αSMA levels in a TGFβ1 dose-dependent manner from NHLFs and IPF-LFs in the co-culture system. Note a stronger αSMA response in IPF-LFs (mean + s.d., n = 3 independent experiments from 6 NHLFs and 6 IPF-LFs donors, ****p < 0.0001, Nonlinear Regression). (f) Schematic workflow of the substitution system in which EPCs were subjected to TGFβ1 stimulation for 3 h in co-culture with NHLFs, after which (1) NHLFs or (2) EPCs were replaced with untreated counterparts or (3) both fresh medium and untreated NHLFs for a further 72 h. (g, h) 72 h after substitutions in the co-culture, αSMA and E-cad levels of NHLFs and EPCs, respectively, were measured with ELISA and normalised to the control. Note replacement of injured EPCs with untreated EPCs or replacement of media and NHLFs attenuates αSMA expression and increases E-cad levels (mean + s.d., ***p < 0.001, **p < 0.01, *p < 0.05, n = 3 independent experiments with 6 NHLFs donors, ANOVA/Dunnett’s). (i) Hierarchical clustering of differentially regulated transcripts from RNA-seq between vehicle and TGFβ1-treated EPCs in the co-culture (p-adj < 0.05). Significantly regulated pro-fibrotic genes (marked by asterisk) are listed on the right side. (j) Volcano plot representing logarithmic ratio of differentially secreted proteins from proteomics analysis of co-culture medium upon apical TGFβ1 stimulation (p-adj < 0.05), with examples of pro-fibrotic proteins. (k-m) Gene Ontology (GO) analysis of differentially expressed genes (p-adj < 0.05) that are enriched in TGFβ1-treated (k), vehicle treated (l) EPCs, and differentially secreted proteins (m). (n, o) GSEA shows enrichment of genes that are known targets of Polycomb complexes (defined by SUZ12, BMI1 and EZH2) upon TGFβ1 treatment in the co-culture system. (p) GSEA shows enrichment of an IPF whole lung signature in TGFβ1-treated EPCs in the co-culture system.

Article Snippet: Where indicated, cells were treated with ROCK inhibitor (Y27632, Sigma-Aldrich, 10 µM), EZH2 inhibitor (GSK126, 10μnM) and TGFβ1 (R&D, 240-B, 1 ng/ml).

Techniques: Co-Culture Assay, Immunofluorescence, Enzyme-linked Immunosorbent Assay, Staining, Expressing, RNA Sequencing Assay

Journal: bioRxiv

Article Title: A polycomb-independent role of EZH2 in TGFβ1-damaged epithelium triggers a fibrotic cascade with mesenchymal cells

doi: 10.1101/2020.07.29.225300

Figure Lengend Snippet: (a) 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 EPCs from co-culture with LFs (n = 5 independent experiments with >50 cells per experiement, scale bars 50 m, *p = 0.0625, Wilcoxon test). (b) ChIP-qPCR shows decreased occupancy of H3K27me3 at promoters of pro-fibrotic genes in EPCs subjected to apical TGFβ1 for 72 h. Unspecific IgG was used as a negative control (mean + s.d., n = 3). (c) ChIP-qPCR shows increased EZH2 occupancy at promoters of profibrotic genes in EPCs subjected to TGFβ1 for 72 h (mean +s.d., n= 3). (d) Representative western blot analysis of ph-EZH2 and quantification shows increased ph-EZH2 levels at T311 and T487 in EPCs subjected to apical TGFβ1 for 72 h (n = 5 independent experiments, mean + s.d., **p = 0.0094, ***p = 0.0008, two-sided unpaired t-test). (e) Representative western blot analysis of SUZ12 immunoprecipitates shows co-precipitation of EZH1 and EZH2 in EPCs. TGFβ1 treatment leads to the EZ-switch from SUZ12-bound EZH2 to EZH1. Unspecific IgG binding was used as a negative control. A representative from 3 independent experiments is shown. (f) ChIP-qPCR shows increased ph-EZH2 occupancy at promoters of pro-fibrotic genes in EPCs subjected to TGFβ1 for 72 h. Note no changes in ph-EZH2 levels at promoters of non-target genes (mean + s.d., n = 3). (g) Increased EZH1 occupancy at promoters of non-fibrotic genes in EPCs subjected to apical TGFβ1 for 72 h (mean +s.d., n = 3). (h) No changes in H3K27me3 at promoters of non-fibrotic genes in EPCs subjected to apical TGFβ1 for 72 h (mean + s.d., n = 3).

Article Snippet: Where indicated, cells were treated with ROCK inhibitor (Y27632, Sigma-Aldrich, 10 µM), EZH2 inhibitor (GSK126, 10μnM) and TGFβ1 (R&D, 240-B, 1 ng/ml).

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

Journal: bioRxiv

Article Title: A polycomb-independent role of EZH2 in TGFβ1-damaged epithelium triggers a fibrotic cascade with mesenchymal cells

doi: 10.1101/2020.07.29.225300

Figure Lengend Snippet: Representative ph-EZH2 images of a confocal plane at the level of the nucleus from vehicle and TGFβ1-treated EPCs (scale bars 7.5μm). Right side panels show linescans through the cytoplasm and the nucleus indicating enrichment of ph-EZH2 in both compartments upon TGFβ1 stimulation. Representation from 3 independent experiments is shown.

Article Snippet: Where indicated, cells were treated with ROCK inhibitor (Y27632, Sigma-Aldrich, 10 µM), EZH2 inhibitor (GSK126, 10μnM) and TGFβ1 (R&D, 240-B, 1 ng/ml).

Techniques:

Journal: bioRxiv

Article Title: A polycomb-independent role of EZH2 in TGFβ1-damaged epithelium triggers a fibrotic cascade with mesenchymal cells

doi: 10.1101/2020.07.29.225300

Figure Lengend Snippet: (a) Representative western blot analysis and quantification show a reduction in ph-EZH2 levels in EZH2-depleted EPCs versus control cells upon TGFβ1 stimulation for 72 h (n = 4 independent experiments, mean + s.d., *p < 0.05, Kruskal-Wallis/Dunn’s). (b) qPCR analysis of pro-fibrotic/EMT target genes in EZH2-depleted EPCs in co-culture with NHLFs shows that EZH2 is required for the effect of TGFβ1 on the upregulation of pro-fibrotic/EMT target genes (n = 4 independent experiments with 4 NHLFs donors, mean + s.d., ****p < 0.001, *p < 0.05, n = 4 independent experiments, ANOVA/Tukey’s). (c) ELISA analysis of signature FMT markers from NHLFs in the co-culture system shows that EHZ2-depleted EPCs prevent the effect of TGFβ1on the epithelial secretions driven FMT process (n = 4 NHLFs donors, mean + s.d., ****p < 0.001, **p = 0.004, *p = 0.0459, ANOVA/Tukey’s). (d) Representative western blot analysis of EZH2 co-immunoprecipitates shows increased levels of EZH2-bound POL2, ph-EHZ2, and actin in TGFβ1-treated EPCs. Unspecific IgG binding was used as a negative control. A representative from 3 independent experiments is shown. (e) Representative western blot analysis of POL2 immunoprecipitates shows co-precipitation of ph-EZH2 and nuclear actin. Increased levels of POL2-bound ph-EZH2 and nuclear actin are observed upon in EPCs subjected to apical TGFβ1 for 72 h. Unspecific IgG binding was used as a negative control. (f) ChIP-qPCR shows increased occupancy of POL2(S5p) at promoters of pro-fibrotic genes in EPCs subjected to TGFβ1 for 24 h. Negative IgG control is shown in Supplementary Fig. 3 (n = 3 independent experiments). (g) ChIP-qPCR shows increased occupancy of POL2(S2p) at pro-fibrotic genes in EPCs subjected to TGFβ1 for 48 h. Negative IgG control is shown in Supplementary Fig. 3 (n = 3 independent experiments).

Article Snippet: Where indicated, cells were treated with ROCK inhibitor (Y27632, Sigma-Aldrich, 10 µM), EZH2 inhibitor (GSK126, 10μnM) and TGFβ1 (R&D, 240-B, 1 ng/ml).

Techniques: Western Blot, Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Binding Assay, Negative Control

Journal: bioRxiv

Article Title: A polycomb-independent role of EZH2 in TGFβ1-damaged epithelium triggers a fibrotic cascade with mesenchymal cells

doi: 10.1101/2020.07.29.225300

Figure Lengend Snippet: (a) Representative F-actin (phalloidin) and DAPI images of EPCs show that treatment with ROCK inhibitor Y27632 but not depletion of EZH2 can prevent TGFβ1-induced actomyosin remodelling in EPCs (scale bars 200μm). (b) Representative western blot analysis and quantification show that blocking actomyosin remodelling by Y27632 prevents TGFβ1-induced ph-EHZ2 in EPCs (n = 3 independent experiments, *p = 0.0523, Kruskal-Wallis/Dunn’s). (c) Representative immunoprecipates of EHZ2 shows abolition of TGFβ1-induced pro-fibrotic transcriptional complex of phEZH2-POL2-actin upon the convergent treatment of TGFβ1 and Y27632. Unspecific IgG binding was used as a negative control. Representative from 3 independent experiments is shown. (d) ELISA analysis of E-cad levels from EPCs in mono- or co-culture with NHLFs and IPF-LFs subjected to TGFβ1 for 72 h shows that blocking actomyosin remodelling by Y27632 prevents TGFβ1-reduced E-cad (n = 3 independent experiments with 6 NHLFs and 6 IPF-LFs donors, mean + s.d., ***p < 0.001, ANOVA/Tukey’s). (e) ELISA analysis of αSMA levels from NHLFs and IPF-LFs in mono and co-culture with EPCs shows that blocking actomyosin remodelling in EPCs leads to the abolition of epithelial secretions that drive increased αSMA upon TGFβ1 stimulation (n = 3 independent experiments with 6 NHLFs and 6 IPF-LFs donors, mean + s.d., ***p < 0.001, ANOVA/Tukey’s).

Article Snippet: Where indicated, cells were treated with ROCK inhibitor (Y27632, Sigma-Aldrich, 10 µM), EZH2 inhibitor (GSK126, 10μnM) and TGFβ1 (R&D, 240-B, 1 ng/ml).

Techniques: Western Blot, Blocking Assay, Binding Assay, Negative Control, Enzyme-linked Immunosorbent Assay, Co-Culture Assay

Journal: bioRxiv

Article Title: A polycomb-independent role of EZH2 in TGFβ1-damaged epithelium triggers a fibrotic cascade with mesenchymal cells

doi: 10.1101/2020.07.29.225300

Figure Lengend Snippet: (a) Schematic workflow of chronic TGFβ1 treatment during SAECs stratification and differentiation, followed by co-culture with LFs. (b) Representative images showing morphological changes of SAECs upon chronic TGFβ1 treatment (scale bars 500μm). (c) Analysis of cilia beat frequency and cilia area shows no significant differences between nSAECs and tSAECs (n = 3 donors, mean + s.d., ns = non-significant, paired t-test) (d) ELISA analysis shows increased pro-fibrotic proteins secretion in tSAECs (n = 3 independent experiments from 4 donors, *p = 0.0313, **p = 0.0078, ***p = 0.0002, paired Wilcoxon test, box plots show minimum, first quartile, median, third quartile and maximum). (e) ELISA analysis of pro-fibrotic markers in the mono- and co-culture system between SAECs and LFs. Note elevated pro-fibrotic levels in tSAECs and the co-culture system. (n = 3 independent experiments from 4 SAECs donors and 6 LFs donors, box plots show minimum, first quartile, median, third quartile and maximum). (f) Western blot analysis shows reduced ph-EZH2 levels upon GSK126 treatment in tSAECs.

Article Snippet: Where indicated, cells were treated with ROCK inhibitor (Y27632, Sigma-Aldrich, 10 µM), EZH2 inhibitor (GSK126, 10μnM) and TGFβ1 (R&D, 240-B, 1 ng/ml).

Techniques: Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: bioRxiv

Article Title: A polycomb-independent role of EZH2 in TGFβ1-damaged epithelium triggers a fibrotic cascade with mesenchymal cells

doi: 10.1101/2020.07.29.225300

Figure Lengend Snippet: (a) Haematoxylin-eosin (H&E) staining of stratified and differentiated SAECs (scale bars 50μm). (b) Nanoindentation shows increased stiffness in tSAECs. Box plots display minimum, first quartile, median, third quartile and maximum (n = 5 SAECs donors, *p = 0.0179, two-sided paired t-test). (c) FITC dextran permeability assay reveals loss of epithelial integrity in tSAECs (n = 4 SAECs donors, mean ± s.d., ****p < 0.0001, Linear Regression). (d) ELISA analysis of αSMA levels from NHLFs in mono-(black line) or co-culture with nSAECs (blue) or tSAECs (red) shows an anti-fibrotic effect on nSAECs subjected to apical TGFβ1 for 72 h (n = 5 SAECs and 5 NHLFs donors,*p < 0.05, **p = 0.01, 2-way ANOVA /Tukey’s). (e) Representative western blot analysis of EZH2 immunoprecipitates shows co-precipitation of ph-EZH2, POL2(S2p) and actin in tSAECs. Note the loss of EZH2-bound SUZ12 in tSAECs compared to nSAECs. Unspecific IgG was used as a negative control. (f) ELISA analysis shows decreased E-cad levels in tSAECs in the mono- or co-culture system. This reduction can be rescued by the ROCK inhibitor Y27632 (n = 4 SAECs donors, mean + s.d., *p < 0.05, **p = 0.0052, 2-way ANOVA/Tukey’s). (g) ELISA analysis of aSMA levels shows increased aSMA levels in NHLFs when co-cultured with tSAECs. Blocking actomyosin remodelling by Y27632 can prevent this effect (n = 4 NHLFs donors, mean + s.d., *p < 0.05, 2-way ANOVA/Tukey’s). (h) Representative western blot analysis and quantification show increased ph-EZH2 levels in Bleomycin treated mice compared to saline control group (n = 10 mice / treatment, mean + s.d., *p = 0.04, ****p < 0.001, Mann-Whitney). (i) A model describing TGFβ1-injured epithelium initiating a bi-directional fibrotic crosstalk with fibroblasts. TGFβ1-injured epithelium promotes (1) an EZ-switch from EZH2-PRC2 to EZH1-PRC1, which is required to maintain H3K27me3 at TGFβ1 non-target genes; and (2) a PRC2-independent EZH2 that forms a pro-fibrotic transcriptional complex with POL2(S2p) and nuclear actin to fine-tune transcription at pro-fibrotic genes.

Article Snippet: Where indicated, cells were treated with ROCK inhibitor (Y27632, Sigma-Aldrich, 10 µM), EZH2 inhibitor (GSK126, 10μnM) and TGFβ1 (R&D, 240-B, 1 ng/ml).

Techniques: Staining, FITC-Dextran Permeability Assay, Enzyme-linked Immunosorbent Assay, Co-Culture Assay, Western Blot, Negative Control, Cell Culture, Blocking Assay, MANN-WHITNEY

Journal: Advanced Science

Article Title: Yin Yang 1‐Induced Long Noncoding RNA DUXAP9 Drives the Progression of Oral Squamous Cell Carcinoma by Blocking CDK1‐Mediated EZH2 Degradation

doi: 10.1002/advs.202207549

Figure Lengend Snippet: DUXAP9 promoted xenograft tumor growth and metastasis of OSCC cells. A,B) The volumes and weights A) and the growth curves B) of tumors derived from CAL27 cells transfected with SS‐NC or SS‐DUXAP9 ASO were measured, and representative tumor images were taken. n = 6/group. C,D) The volumes and weights C) and the growth curves D) of tumors derived from CAL27 cells transfected with vector or DUXAP9 plasmids were measured, and representative tumor images were taken. n = 6/group. E) Immunohistochemical staining of Ki67, PCNA, EZH2, and E‐cadherin in tumors derived from CAL27 cells transfected with SS‐NC and SS‐DUXAP9 ASO or control‐ and DUXAP9‐expressing vectors, respectively, Scale bars, 100 µm (left), 25 µm (right). F–I) The expression of Ki67 F), PCNA G), EZH2 H), and E‐cadherin I) was determined by the IHC score in the indicated groups. J) Representative bioluminescence images of lung metastasis in mice injected with CAL27 cells stably expressing vector or DUXAP9 via the tail vein. K) Representative images of H&E staining and GFP fluorescence in the lungs of nude mice injected with CAL27 cells overexpressing vector or DUXAP9. Scale bars, 100 µm. Data in (B) and (D) are presented as the mean ± SEM, and data in (A), (C), and (F–I) are presented as the mean ± SD from three independent experiments. Data in (A–D) and (F–I) were calculated by two‐tailed unpaired Student's t ‐test.

Article Snippet: An in vitro reconstituted RIP assay was conducted with human recombinant EZH2 protein (#TP302054, Origene, US) and DUXAP9 transcribed by a T7 High Yield RNA Transcription Kit (Beyotime, China).

Techniques: Derivative Assay, Transfection, Plasmid Preparation, Immunohistochemical staining, Staining, Control, Expressing, Injection, Stable Transfection, Fluorescence, Two Tailed Test

Journal: Advanced Science

Article Title: Yin Yang 1‐Induced Long Noncoding RNA DUXAP9 Drives the Progression of Oral Squamous Cell Carcinoma by Blocking CDK1‐Mediated EZH2 Degradation

doi: 10.1002/advs.202207549

Figure Lengend Snippet: Physical interaction between DUXAP9 and EZH2 in OSCC cells. A) DUXAP9‐interacting proteins were separated by SDS‐PAGE followed by an endogenous DUXAP9 RNA pull‐down assay and manifested by Coomassie Brilliant Blue staining. The distinct protein bands in the gel were excised, dissolved, and subjected to mass spectrometry. The red asterisk denotes the location of EZH2. B) Venn diagram shows a group of nucleoplasm‐located proteins identified by proteomic analysis. The five proteins listed are considered candidates for DUXAP9 binding proteins. C) RIP‐qPCR assay using IgG or anti‐EZH2 antibody shows the enrichment of DUXAP9 expression in the precipitated EZH2 binding complex. D) RNA pull‐down assays with biotin‐labeled DUXAP9 probes show the interaction between DUXAP9 and EZH2 in CAL27 cells. E) Confocal FISH and IF images showing the colocalization of EZH2 (green) and DUXAP9 (red) in CAL27 and HN6 cells. Scale bars, 5 µm. F) Predicted DUXAP9 interaction region using catRAPID. G) qRT‐PCR analysis of DUXAP9 enrichment by RIP assay using anti‐EZH2 in CAL27 cells. Thirteen specific primers for DUXAP9 were used to detect the binding region of DUXAP9. H) Predicted secondary structure of DUXAP9 using Mfold software. I) Western blot followed by RNA pull‐down assay of DUXAP9‐ or mutant DUXAP9 (mut‐DUXAP9)‐transfected cells. The construction of the mutant DUXAP9 vector is shown above, and the expression level of DUXAP9‐associated EZH2 protein is shown below. J) In vitro reconstituted RIP‐qPCR assay using IgG or EZH2 antibody shows the enrichment of DUXAP9 expression in the precipitated EZH2 binding complex. Data are presented as the mean ± SD from three independent experiments. Data in (C), (G), and (J) were calculated by two‐tailed unpaired Student's t ‐test.

Article Snippet: An in vitro reconstituted RIP assay was conducted with human recombinant EZH2 protein (#TP302054, Origene, US) and DUXAP9 transcribed by a T7 High Yield RNA Transcription Kit (Beyotime, China).

Techniques: SDS Page, Pull Down Assay, Staining, Mass Spectrometry, Binding Assay, Expressing, Labeling, Quantitative RT-PCR, Software, Western Blot, Mutagenesis, Transfection, Plasmid Preparation, In Vitro, Two Tailed Test

Journal: Advanced Science

Article Title: Yin Yang 1‐Induced Long Noncoding RNA DUXAP9 Drives the Progression of Oral Squamous Cell Carcinoma by Blocking CDK1‐Mediated EZH2 Degradation

doi: 10.1002/advs.202207549

Figure Lengend Snippet: DUXAP9 increases EZH2 protein expression via inhibition of its proteasomal degradation. A) The mRNA expression of EZH2 was measured by qRT‐PCR in CAL27 and HN6 cells transfected with SS‐NC or SS‐DUXAP9 ASO (left) or control‐ or DUXAP9‐overexpressing vectors (right). B) The protein expression of EZH2 was detected by western blot in CAL27 and HN6 cells transfected with SS‐NC or SS‐DUXAP9 ASO (left) or control or DUXAP9 overexpressing vectors (right). C–E) Western blot shows EZH2 protein in CAL27 and HN6 cells transfected with SS‐NC or SS‐DUXAP9 ASO C) or control‐ or wild type DUXAP9‐ D) or mutant DUXAP9‐ E) overexpressing vectors and treated with CHX (20 µg mL −1 ) for the indicated time (left). The quantification of the EZH2 degradation rate was measured by grayscale analysis (right). F) Western blot showing EZH2 protein in CAL27 and HN6 cells transfected with SS‐NC or SS‐DUXAP9 and treated with MG132 (20 µ m for 6 h). G, H) Ubiquitination of EZH2 in DUXAP9‐silenced G) and DUXAP9‐overexpressing H) CAL27 cells after MG132 treatment (20 µ m for 6 h) was detected by western blot analysis. I) Ubiquitination of EZH2 in 293T cells transfected with EZH2 and DUXAP9 after MG132 treatment (20 µ m for 6 h) was detected by western blot analysis. Data are presented as the mean ± SD from three independent experiments. Data in (A) and (C–E) were calculated by two‐tailed unpaired Student's t ‐test.

Article Snippet: An in vitro reconstituted RIP assay was conducted with human recombinant EZH2 protein (#TP302054, Origene, US) and DUXAP9 transcribed by a T7 High Yield RNA Transcription Kit (Beyotime, China).

Techniques: Expressing, Inhibition, Quantitative RT-PCR, Transfection, Control, Western Blot, Mutagenesis, Two Tailed Test

Journal: Advanced Science

Article Title: Yin Yang 1‐Induced Long Noncoding RNA DUXAP9 Drives the Progression of Oral Squamous Cell Carcinoma by Blocking CDK1‐Mediated EZH2 Degradation

doi: 10.1002/advs.202207549

Figure Lengend Snippet: DUXAP9 suppresses EZH2 degradation via inhibition of the phosphorylation (Thr345/Thr487) of EZH2. A,B) Western blot showing the levels of p‐EZH2 (T345), p‐EZH2 (T487), and CDK1 in DUXAP9‐silenced A) and DUXAP9‐overexpressing B) CAL27 and HN6 cells treated with MG132 (20 µ m for 6 h). C,D) Western blot showing the interaction between EZH2 and CDK1 in control and DUXAP9‐overexpressing CAL27 C) and 293T D) cells treated with MG132 (20 µ m for 6 h) in a coimmunoprecipitation assay using an anti‐EZH2 antibody. E,F) The binding of DUXAP9 to mutant EZH2 or wild‐type EZH2 was detected by RNA pull‐down assay E) and RIP‐qPCR assay F). The diagram shows the mutation site of EZH2. G,H) Western blot showing the levels of p‐EZH2 (T345), p‐EZH2 (T487), and CDK1 in the different combination of transfection of DUXAP9‐ and CDK1 overexpressing vectors in CAL27 G) and 293T H) cells transfected with different combinations of DUXAP9‐ and CDK1‐overexpressing vectors and treated with MG132 (20 µ m for 6 h). I,J) Western blot showing the ubiquitination of EZH2 in the different combination of transfection of DUXAP9‐ and CDK1 overexpressing vectors in CAL27 I) and 293T J) cells transfected with different combinations of DUXAP9‐ and CDK1‐overexpressing vectors and treated with MG132 (20 µ m for 6 h). K) Western blot shows the ubiquitination of mutant EZH2 in the different combination of transfection of DUXAP9‐ and CDK1 overexpressing vectors in 293T cells transfected with different combinations of DUXAP9‐ and CDK1‐overexpressing vectors and treated with MG132 (20 µ m for 6 h). L) Western blot showing the levels of p‐EZH2 (T345), p‐EZH2 (T487), and CDK1 in the different combinations of mutant DUXAP9‐ and CDK1‐overexpressing vectors treated with MG132 (20 µ m for 6 h). M) Western blot analysis of the ubiquitination of EZH2 in 293T cells transfected with different combinations of mutant DUXAP9‐ and CDK1‐overexpressing vectors and treated with MG132 (20 µ m for 6 h). Data are presented as the mean ± SD from three independent experiments. Data in (F) were calculated by two‐tailed unpaired Student's t ‐test.

Article Snippet: An in vitro reconstituted RIP assay was conducted with human recombinant EZH2 protein (#TP302054, Origene, US) and DUXAP9 transcribed by a T7 High Yield RNA Transcription Kit (Beyotime, China).

Techniques: Inhibition, Western Blot, Control, Co-Immunoprecipitation Assay, Binding Assay, Mutagenesis, Pull Down Assay, Transfection, Two Tailed Test

Journal: Advanced Science

Article Title: Yin Yang 1‐Induced Long Noncoding RNA DUXAP9 Drives the Progression of Oral Squamous Cell Carcinoma by Blocking CDK1‐Mediated EZH2 Degradation

doi: 10.1002/advs.202207549

Figure Lengend Snippet: DUXAP9 suppresses EZH2 degradation via nuclear‐to‐cytoplasmic translocation. A–C) Western blot showing the levels of nuclear and cytoplasmic EZH2 in CDK1‐ A) and DUXAP9‐ B) overexpressing and DUXAP9 knockdown C) CAL27 and HN6 cells. Quantification of the EZH2 nucleus/cytoplasm ratio by grayscale analysis is shown on the right. D) Western blot shows the levels of nuclear and cytoplasmic mutant EZH2 in vector‐ or CDK1‐overexpressing 293T cells. E) Western blot shows the levels of nuclear and cytoplasmic wild‐type EZH2 and mutant EZH2 in CDK1‐overexpressing 293T cells. F) Western blot shows the levels of nuclear and cytoplasmic EZH2 in 293T cells transfected with control or mutant DUXAP9‐overexpressing vectors in combination with CDK1‐overexpressing vector. Data are presented as the mean ± SD from three independent experiments. Data in (A–F) were calculated by two‐tailed unpaired Student's t ‐test.

Article Snippet: An in vitro reconstituted RIP assay was conducted with human recombinant EZH2 protein (#TP302054, Origene, US) and DUXAP9 transcribed by a T7 High Yield RNA Transcription Kit (Beyotime, China).

Techniques: Translocation Assay, Western Blot, Knockdown, Mutagenesis, Plasmid Preparation, Transfection, Control, Two Tailed Test

Journal: Advanced Science

Article Title: Yin Yang 1‐Induced Long Noncoding RNA DUXAP9 Drives the Progression of Oral Squamous Cell Carcinoma by Blocking CDK1‐Mediated EZH2 Degradation

doi: 10.1002/advs.202207549

Figure Lengend Snippet: DUXAP9 promotes the proliferation and invasion of OSCC cells by mediating EZH2 expression and function. A) The mRNA expression of 9 known EZH2 target genes was analyzed by qRT‐PCR assays in CAL27 and HN6 cells transfected with vector, DUXAP9, and EZH2. B) ChIP‐qPCR assay of EZH2 or IgG occupancy at the CDKN1A, DAB2IP, and RUNX2 loci in CAL27 cells transfected with SS‐NC or SS‐DUXAP9. C) The volumes, weights and growth curves of tumors derived from CAL27 cells transfected with control‐ or DUXAP9‐expressing vectors and siRNAs targeting NC or EZH2 were measured and imaged; D,E) The expression of Ki67, PCNA, and EZH2 was determined by the IHC score in the indicated groups. n = 5/group, Scale bars, 100 µm (left), 25 µm (right). Data in (C) are presented as the mean ± SEM, and data in (A,B) and (D) are presented as the mean ± SD from three independent experiments. Data were calculated by two‐tailed unpaired Student's t ‐test.

Article Snippet: An in vitro reconstituted RIP assay was conducted with human recombinant EZH2 protein (#TP302054, Origene, US) and DUXAP9 transcribed by a T7 High Yield RNA Transcription Kit (Beyotime, China).

Techniques: Expressing, Quantitative RT-PCR, Transfection, Plasmid Preparation, Derivative Assay, Control, Two Tailed Test

Journal: Advanced Science

Article Title: Yin Yang 1‐Induced Long Noncoding RNA DUXAP9 Drives the Progression of Oral Squamous Cell Carcinoma by Blocking CDK1‐Mediated EZH2 Degradation

doi: 10.1002/advs.202207549

Figure Lengend Snippet: Schematic depicting YY1‐induced DUXAP9 drives OSCC by blocking CDK1‐mediated EZH2 degradation. DUXAP9 orchestrates a different biological function of CDK1‐mediated phosphorylation of the T345 and T487 sites of EZH2 in controlling the protein stability of EZH2 via nuclear to cytoplasmic translocation, suggesting the importance of lncRNA regulation at the posttranslational level in OSCC progression.

Article Snippet: An in vitro reconstituted RIP assay was conducted with human recombinant EZH2 protein (#TP302054, Origene, US) and DUXAP9 transcribed by a T7 High Yield RNA Transcription Kit (Beyotime, China).

Techniques: Blocking Assay, Translocation Assay

Journal: Nature cell biology

Article Title: A PRC2-independent function for EZH2 in regulating rRNA 2′-O methylation and IRES-dependent translation

doi: 10.1038/s41556-021-00653-6

Figure Lengend Snippet: (a, b) C4–2 cells were lysed and subjected to co-IP assay using anti-EZH2 (a) or anti-FBL antibody (b), followed by western blot analysis with indicated antibodies. Rabbit IgG was set as a negative control.

Article Snippet: Purified recombinant human EZH2, FBL and NOP56 proteins ( Supplementary Table 5 ) were purchased from Origene and exchanged into K150 buffer (20 mM KH 2 PO 4 , pH 7.4, 10% glycerol, 150 mM KCl, 0.01% NP40 and 1 mM BME) to avoid non-protein sourced primary amine groups in the crosslinking mixture, which can react with the crosslinking reagent, BS 3 (bis[sulfosuccinimidyl] suberate, Thermo).

Techniques: Co-Immunoprecipitation Assay, Western Blot, Negative Control

Journal: Nature cell biology

Article Title: A PRC2-independent function for EZH2 in regulating rRNA 2′-O methylation and IRES-dependent translation

doi: 10.1038/s41556-021-00653-6

Figure Lengend Snippet: (a) Purified proteins of GST-tagged EED and Flag-tagged FBL were subjected to GST pull down assay, followed by western blot analysis.

Article Snippet: Purified recombinant human EZH2, FBL and NOP56 proteins ( Supplementary Table 5 ) were purchased from Origene and exchanged into K150 buffer (20 mM KH 2 PO 4 , pH 7.4, 10% glycerol, 150 mM KCl, 0.01% NP40 and 1 mM BME) to avoid non-protein sourced primary amine groups in the crosslinking mixture, which can react with the crosslinking reagent, BS 3 (bis[sulfosuccinimidyl] suberate, Thermo).

Techniques: Modification, Purification, Pull Down Assay, Western Blot

Journal: Nature cell biology

Article Title: A PRC2-independent function for EZH2 in regulating rRNA 2′-O methylation and IRES-dependent translation

doi: 10.1038/s41556-021-00653-6

Figure Lengend Snippet: (a) RTL-P assay to detect the 2′-O-Me level in 12 areas in rRNA. Total RNAs were extracted and subjected to reverse transcription (RT) with RT primer at low (1 μM) or high (1 mM) concentration of dNTP, respectively. The obtained cDNA was then amplified with primer pairs corresponding to upstream (Um) or downstream (Dm) regions of specific methylation site(s). This assay has been performed three times with similar results.

Article Snippet: Purified recombinant human EZH2, FBL and NOP56 proteins ( Supplementary Table 5 ) were purchased from Origene and exchanged into K150 buffer (20 mM KH 2 PO 4 , pH 7.4, 10% glycerol, 150 mM KCl, 0.01% NP40 and 1 mM BME) to avoid non-protein sourced primary amine groups in the crosslinking mixture, which can react with the crosslinking reagent, BS 3 (bis[sulfosuccinimidyl] suberate, Thermo).

Techniques: Reverse Transcription, Concentration Assay, Amplification, Methylation

Journal: Nature cell biology

Article Title: A PRC2-independent function for EZH2 in regulating rRNA 2′-O methylation and IRES-dependent translation

doi: 10.1038/s41556-021-00653-6

Figure Lengend Snippet: (a) Western blot analysis of FBL protein level in PCa cell lines upon EZH2 knockdown.

Article Snippet: Purified recombinant human EZH2, FBL and NOP56 proteins ( Supplementary Table 5 ) were purchased from Origene and exchanged into K150 buffer (20 mM KH 2 PO 4 , pH 7.4, 10% glycerol, 150 mM KCl, 0.01% NP40 and 1 mM BME) to avoid non-protein sourced primary amine groups in the crosslinking mixture, which can react with the crosslinking reagent, BS 3 (bis[sulfosuccinimidyl] suberate, Thermo).

Techniques: Binding Assay, Western Blot, Knockdown

Journal: Nature cell biology

Article Title: A PRC2-independent function for EZH2 in regulating rRNA 2′-O methylation and IRES-dependent translation

doi: 10.1038/s41556-021-00653-6

Figure Lengend Snippet: (a) Co-IP of box C/D snoRNP components NOP56, NOP58 and SNU13 with FBL in control and EZH2-deficient C4–2 cells, followed by western blot analysis with indicated antibodies. Graph represents the relative NOP56 protein level coimmunoprecipitated with FBL in each group. Data represent Mean ± SD (n=3 biologically independent measurements). Interaction intensity at control group was set as 1. Statistical significance was determined by two-tailed Student’s t-test.

Article Snippet: Purified recombinant human EZH2, FBL and NOP56 proteins ( Supplementary Table 5 ) were purchased from Origene and exchanged into K150 buffer (20 mM KH 2 PO 4 , pH 7.4, 10% glycerol, 150 mM KCl, 0.01% NP40 and 1 mM BME) to avoid non-protein sourced primary amine groups in the crosslinking mixture, which can react with the crosslinking reagent, BS 3 (bis[sulfosuccinimidyl] suberate, Thermo).

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

Journal: Nature cell biology

Article Title: A PRC2-independent function for EZH2 in regulating rRNA 2′-O methylation and IRES-dependent translation

doi: 10.1038/s41556-021-00653-6

Figure Lengend Snippet: (a) Mixtures of recombinant EZH2, FBL and NOP56 proteins with or without BS3 crosslinking were subjected to SDS-PAGE, followed by western blot analysis using their own antibodies to visualize the location of cross-linked species.

Article Snippet: Purified recombinant human EZH2, FBL and NOP56 proteins ( Supplementary Table 5 ) were purchased from Origene and exchanged into K150 buffer (20 mM KH 2 PO 4 , pH 7.4, 10% glycerol, 150 mM KCl, 0.01% NP40 and 1 mM BME) to avoid non-protein sourced primary amine groups in the crosslinking mixture, which can react with the crosslinking reagent, BS 3 (bis[sulfosuccinimidyl] suberate, Thermo).

Techniques: Recombinant, SDS Page, Western Blot

Journal: Nature cell biology

Article Title: A PRC2-independent function for EZH2 in regulating rRNA 2′-O methylation and IRES-dependent translation

doi: 10.1038/s41556-021-00653-6

Figure Lengend Snippet: (a) Nuclear extracts from control and EZH2-deficient C4–2 cells were subjected to size-exclusion and the protein levels of FBL, NOP56, NOP58 and SNU13 were determined by western blot analysis in all samples. Protein distributions of EZH2 and SUZ12 in control cells were also detected as references. This assay has been performed three times with similar results.

Article Snippet: Purified recombinant human EZH2, FBL and NOP56 proteins ( Supplementary Table 5 ) were purchased from Origene and exchanged into K150 buffer (20 mM KH 2 PO 4 , pH 7.4, 10% glycerol, 150 mM KCl, 0.01% NP40 and 1 mM BME) to avoid non-protein sourced primary amine groups in the crosslinking mixture, which can react with the crosslinking reagent, BS 3 (bis[sulfosuccinimidyl] suberate, Thermo).

Techniques: Control, Western Blot

Journal: Nature cell biology

Article Title: A PRC2-independent function for EZH2 in regulating rRNA 2′-O methylation and IRES-dependent translation

doi: 10.1038/s41556-021-00653-6

Figure Lengend Snippet: (a, b) Comparison of FBL (a) or NOP56 (b) mRNA levels in PCa patient samples with different Gleason grades using JHMI cohort. The ends of the box are the upper and lower quartiles and the box spans the interquartile range. The median is marked by a vertical line inside the box and the whiskers represent for 1.5x interquartile range. The significance of trend was calculated by two-sided Kruskal-Wallis test. ‘n’ represents the number of patients included in the analyses.

Article Snippet: Purified recombinant human EZH2, FBL and NOP56 proteins ( Supplementary Table 5 ) were purchased from Origene and exchanged into K150 buffer (20 mM KH 2 PO 4 , pH 7.4, 10% glycerol, 150 mM KCl, 0.01% NP40 and 1 mM BME) to avoid non-protein sourced primary amine groups in the crosslinking mixture, which can react with the crosslinking reagent, BS 3 (bis[sulfosuccinimidyl] suberate, Thermo).

Techniques: Comparison

Journal: Nature cell biology

Article Title: A PRC2-independent function for EZH2 in regulating rRNA 2′-O methylation and IRES-dependent translation

doi: 10.1038/s41556-021-00653-6

Figure Lengend Snippet: (a) Wound healing assay was conducted to evaluate the migration potential of PC-3 cells after FBL depletion. The healing of wounded cell layer was monitored under a microscope every 24 h. Graph showing the rate of filling of the scratched area by cells. Data represent Mean ± SD from n=3 biologically independent experiments. The knockdown efficiency of FBL was validated by western blot.

Article Snippet: Purified recombinant human EZH2, FBL and NOP56 proteins ( Supplementary Table 5 ) were purchased from Origene and exchanged into K150 buffer (20 mM KH 2 PO 4 , pH 7.4, 10% glycerol, 150 mM KCl, 0.01% NP40 and 1 mM BME) to avoid non-protein sourced primary amine groups in the crosslinking mixture, which can react with the crosslinking reagent, BS 3 (bis[sulfosuccinimidyl] suberate, Thermo).

Techniques: Wound Healing Assay, Migration, Microscopy, Knockdown, Western Blot

Journal: Nature cell biology

Article Title: A PRC2-independent function for EZH2 in regulating rRNA 2′-O methylation and IRES-dependent translation

doi: 10.1038/s41556-021-00653-6

Figure Lengend Snippet: EZH2 plays a dual-role to regulate gene expression. On one hand, EZH2 inhibits DNA transcription by catalyzing H3K27me3 marks in a PRC2-dependent manner; On the other hand, EZH2 activates mRNA translation by enhancing the functionality of FBL through a non-lysine methyltransferase role. Hence, EZH2 could exert its oncogenic functions by coordination of transcriptional inhibition (i.e., tumor suppressors) and promotion of translation (i.e., pro-oncogenic, anti-apoptotic, and survival proteins) during cancer progression.

Article Snippet: Purified recombinant human EZH2, FBL and NOP56 proteins ( Supplementary Table 5 ) were purchased from Origene and exchanged into K150 buffer (20 mM KH 2 PO 4 , pH 7.4, 10% glycerol, 150 mM KCl, 0.01% NP40 and 1 mM BME) to avoid non-protein sourced primary amine groups in the crosslinking mixture, which can react with the crosslinking reagent, BS 3 (bis[sulfosuccinimidyl] suberate, Thermo).

Techniques: Control, Gene Expression, Inhibition

Journal: Human Mutation

Article Title: Weaver Syndrome‐Associated EZH2 Protein Variants Show Impaired Histone Methyltransferase Function In Vitro

doi: 10.1002/humu.22946

Figure Lengend Snippet: Weaver syndrome mutants are impaired in their histone methyltransferase activity in vitro . Histone methyltransferase reactions were performed using 2 μg purified core histones and 0.67 μM 3 H‐S‐adenosyl‐methionine ( 3 H‐SAM). Each reaction was incubated with 250 ng of either wild‐type (WT) or a mutant HMTase complex (or no enzyme controls). Histone methyltransferase activity was measured based on the incorporation of 3 H‐labeled methyl groups, represented in scintillation counts per minute. Counts were normalized by subtracting background counts (i.e., no enzyme) from the total counts. A : Incorporation of tritiated methyl groups from 3 H‐SAM onto core histones is shown for each complex: EZH2 WT • , p.(Phe672Ile) × , p.(Pro132Ser) ★ , p.(Tyr153del) △, p.(His694Tyr) ▽, p.(Glu745Lys) ▴, p.(Ala682Thr) ▾, p.(Arg684Cys) ▪, p.(Tyr133Cys) □, and p.(Asp185His) ◇. Error bars represent standard deviation (SD) within the groups “EZH2 WT” and “EZH2 mutants.” Unpaired t‐test showed statistically significant difference between the two groups (P value < 0.0001). B : Incorporation of tritiated methyl groups from 3 H‐SAM onto core histones is shown for the positive control EZH2 WT, the negative control EZH2 (p.Phe672Ile), and the mutant complex with activity closest to WT, namely, EZH2 (p.Pro132Ser). Error bars represent SD of four independent replicates for the controls, and three independent replicates for the mutant EZH2 (p.Pro132Ser). One‐way ANOVA showed statistically significant difference between all groups (overall P value < 0.0001; P values between WT and p.(Phe672Ile), between p.(Phe672Ile) and p.(Pro132Ser), and between WT and p.(Pro132Ser) were all <0.05).

Article Snippet: To test our hypothesis, we designed recombinant human EZH2 proteins, had them preassembled into PRC2 complexes (BPS Bioscience, San Diego CA), and tested their activity in vitro using a well‐accepted in vitro assay [Ernst et al., ; Yap et al., ; Score et al., ].

Techniques: Activity Assay, In Vitro, Purification, Incubation, Mutagenesis, Labeling, Standard Deviation, Positive Control, Negative Control