Journal: Journal of Cell Science

Article Title: Interplay between nuclear survivin and the PRC2 complex and its impact on H3K27me3-directed transcriptional repression

doi: 10.1242/jcs.264572

Figure Lengend Snippet: Hypoxia increases expression of EZH2, H3K27me3 and survivin. (A) Immunoblots of WCEs from U2OS, HeLa and MRC5 lines cultured under normoxic or hypoxic environments (24 h). Blots were immunoprobed with anti-EZH2, anti-H3K27me3 and anti-survivin antibodies. Anti-Hif1a used to prove the hypoxic state had been induced, and anti-tubulin was used as a loading control. (B–D) Quantification of immunoblots represented in A from three independent experiments demonstrating that EZH2, H3K27me3 and survivin are all more abundant under hypoxia. Data presented are means±s.d. * P <0.05, ** P <0.01, *** P <0.001 (two-way ANOVA with Tukey's multiple comparisons post test).

Article Snippet: Primary antibodies were diluted 1:1000 in TBST with 5% milk, unless otherwise stated, and were against: tubulin (Sigma, B512, T5168), β-actin (Invitrogen MA1-140), TBP (CST, 8515), survivin (C60, CST 71G4B7, TBST 2% milk; or 6E4), H3K27me3 (Abcam, ab192985; TBST-2% BSA), GST (Cytivia, RPN1236V), EZH2 (CST, D269 or Proteintech 21800-1-AP), Hif1α (Novus Biologics, NB100-449).

Techniques: Expressing, Western Blot, Cell Culture, Control

Journal: Journal of Cell Science

Article Title: Interplay between nuclear survivin and the PRC2 complex and its impact on H3K27me3-directed transcriptional repression

doi: 10.1242/jcs.264572

Figure Lengend Snippet: Survivin and EZH2 interact. (A) Immunoprecipitation was carried out using whole MRC5 extracts using anti-survivin (C60), anti-EZH2, mouse IgG antibodies (negative control). Co-immunoprecipitation was assessed with the alternative antibodies. Co-immunoprecipitation of EZH2 with survivin was evident when anti-EZH2 was used to immunoprecipitate but not when the anti-survivin (C60) antibody was used. (B) GST pulldown assay was carried out with WCEs prepared from RPE cells expressing GST (negative control), GST–survivin and various GST-tagged survivin truncations, (numbering indicating amino acids), used as bait. (C) Quantification of interactions represented in B. EZH2 binds mainly to the first 90 amino acids of survivin. Data are mean±s.d. from three independent experiments. *** P <0.001; **** P <0.0001; ns, not significant (one-way ANOVA with Dunnett's post hoc test). (D) Immunoprecipitation was carried out as in A but using anti-H3K27me3 specific antibodies, rather than anti-EZH2. Co-immunoprecipitation of survivin and H3K27me3 was evident in reciprocal samples. (E) The GST pulldown experiment as in B was repeated using RPE cell lysates with GST or GST–survivin, and interaction with H3K27me3 determined by immunoblotting. (F) Quantification of data represented in E, normalised to the GST or GST–survivin. Data are mean±s.d., n =3. *** P <0.001 (unpaired two-tailed Student's t -test). Blots in A and D are representative of three repeats. Inputs are 7.5%.

Article Snippet: Primary antibodies were diluted 1:1000 in TBST with 5% milk, unless otherwise stated, and were against: tubulin (Sigma, B512, T5168), β-actin (Invitrogen MA1-140), TBP (CST, 8515), survivin (C60, CST 71G4B7, TBST 2% milk; or 6E4), H3K27me3 (Abcam, ab192985; TBST-2% BSA), GST (Cytivia, RPN1236V), EZH2 (CST, D269 or Proteintech 21800-1-AP), Hif1α (Novus Biologics, NB100-449).

Techniques: Immunoprecipitation, Negative Control, GST Pulldown Assay, Expressing, Western Blot, Two Tailed Test

Journal: Journal of Cell Science

Article Title: Interplay between nuclear survivin and the PRC2 complex and its impact on H3K27me3-directed transcriptional repression

doi: 10.1242/jcs.264572

Figure Lengend Snippet: Survivin knockdown increases H3K27me3 abundance. (A) U2OS and MRC5 cells were incubated with control or survivin-specific siRNA for 48 h. Lysates were immunoblotted with antibodies against the indicated proteins. (B,C) Quantitative analysis of immunoblots, normalised to β-actin loading for (B) U2OS, and (C) MRC5 cells. No change was seen in EZH2 expression but H3K27me3 was increased in both lines. Data are means±s.d. from three independent experiments. * P <0.05; ** P <0.01; *** P <0.001, ns, not significant (two-way ANOVA with Tukey's multiple comparisons post test).

Article Snippet: Primary antibodies were diluted 1:1000 in TBST with 5% milk, unless otherwise stated, and were against: tubulin (Sigma, B512, T5168), β-actin (Invitrogen MA1-140), TBP (CST, 8515), survivin (C60, CST 71G4B7, TBST 2% milk; or 6E4), H3K27me3 (Abcam, ab192985; TBST-2% BSA), GST (Cytivia, RPN1236V), EZH2 (CST, D269 or Proteintech 21800-1-AP), Hif1α (Novus Biologics, NB100-449).

Techniques: Knockdown, Incubation, Control, Western Blot, Expressing

Journal: Journal of Cell Science

Article Title: Interplay between nuclear survivin and the PRC2 complex and its impact on H3K27me3-directed transcriptional repression

doi: 10.1242/jcs.264572

Figure Lengend Snippet: Survivin and EZH2 in ihPSCs. (A) Three pluripotent stem cell lines, CGT-RCIB 10, ReBL Pat and iAT1 were grown in normoxia and immunostained for EZH2 (red), endogenous survivin (green), and counterstained with NucBlue to show the nucleus (blue). Scale bars: 50 µm. (B) There is colocalisation of EZH2 and survivin in the nuclei as shown by the intensity profiles along the yellow line in A (FIJI software). Results representative of N =3 independent repeats. (C) CGT-RCIB 10 cells were incubated with control or survivin-specific siRNA for 24 h. Lysates were immunoblotted with antibodies against the indicated proteins. (D) Quantitative analysis of bands in immunoblots in C, normalised to the β-actin loading control. Although survivin was only partially knocked down, H3K27me3 abundance increased significantly. Data are normalized to control siRNA treatment and are means±s.d. from n =3 plotted. * P <0.05; ** P <0.01; ns, not significant (two-way ANOVA with Tukey's multiple comparisons post test). (E) qPCR analysis was carried out for the genes indicated from CGT-RCIB 10 cells treated with either control or survivin-specific siRNA (24 h). Data are normalized to control siRNA treatment and means±s.d. from N =3 plotted. * P <0.05; ** P <0.01; *** P <0.001; ns, not significant (two-way ANOVA with Tukey's multiple comparisons post test). (F) qPCR analysis of major satellite transcripts from CGT-RCIB 10 cells exposed to control or survivin-specific siRNA. A significant reduction in major satellite expression occurred in the absence of survivin. Data are normalized to control siRNA treatment and means±s.d. from n =3 plotted. ** P <0.01 (unpaired two-tailed Student's t -test).

Article Snippet: Primary antibodies were diluted 1:1000 in TBST with 5% milk, unless otherwise stated, and were against: tubulin (Sigma, B512, T5168), β-actin (Invitrogen MA1-140), TBP (CST, 8515), survivin (C60, CST 71G4B7, TBST 2% milk; or 6E4), H3K27me3 (Abcam, ab192985; TBST-2% BSA), GST (Cytivia, RPN1236V), EZH2 (CST, D269 or Proteintech 21800-1-AP), Hif1α (Novus Biologics, NB100-449).

Techniques: Software, Incubation, Control, Western Blot, Expressing, Two Tailed Test

Journal: Journal of Cell Science

Article Title: Interplay between nuclear survivin and the PRC2 complex and its impact on H3K27me3-directed transcriptional repression

doi: 10.1242/jcs.264572

Figure Lengend Snippet: Hypoxia increases expression of EZH2, H3K27me3 and survivin. (A) Immunoblots of WCEs from U2OS, HeLa and MRC5 lines cultured under normoxic or hypoxic environments (24 h). Blots were immunoprobed with anti-EZH2, anti-H3K27me3 and anti-survivin antibodies. Anti-Hif1a used to prove the hypoxic state had been induced, and anti-tubulin was used as a loading control. (B–D) Quantification of immunoblots represented in A from three independent experiments demonstrating that EZH2, H3K27me3 and survivin are all more abundant under hypoxia. Data presented are means±s.d. * P <0.05, ** P <0.01, *** P <0.001 (two-way ANOVA with Tukey's multiple comparisons post test).

Article Snippet: The following primary antibodies were used at 1:200: anti-survivin (Cell Signaling Technologies 71G4B7 or 6E4), anti-H3K27Me3 (Abcam, Ab192985 ), anti-EZH2 [Cell Signalling Technologies (CST), D269].

Techniques: Expressing, Western Blot, Cell Culture, Control

Journal: Journal of Cell Science

Article Title: Interplay between nuclear survivin and the PRC2 complex and its impact on H3K27me3-directed transcriptional repression

doi: 10.1242/jcs.264572

Figure Lengend Snippet: Survivin and EZH2 interact. (A) Immunoprecipitation was carried out using whole MRC5 extracts using anti-survivin (C60), anti-EZH2, mouse IgG antibodies (negative control). Co-immunoprecipitation was assessed with the alternative antibodies. Co-immunoprecipitation of EZH2 with survivin was evident when anti-EZH2 was used to immunoprecipitate but not when the anti-survivin (C60) antibody was used. (B) GST pulldown assay was carried out with WCEs prepared from RPE cells expressing GST (negative control), GST–survivin and various GST-tagged survivin truncations, (numbering indicating amino acids), used as bait. (C) Quantification of interactions represented in B. EZH2 binds mainly to the first 90 amino acids of survivin. Data are mean±s.d. from three independent experiments. *** P <0.001; **** P <0.0001; ns, not significant (one-way ANOVA with Dunnett's post hoc test). (D) Immunoprecipitation was carried out as in A but using anti-H3K27me3 specific antibodies, rather than anti-EZH2. Co-immunoprecipitation of survivin and H3K27me3 was evident in reciprocal samples. (E) The GST pulldown experiment as in B was repeated using RPE cell lysates with GST or GST–survivin, and interaction with H3K27me3 determined by immunoblotting. (F) Quantification of data represented in E, normalised to the GST or GST–survivin. Data are mean±s.d., n =3. *** P <0.001 (unpaired two-tailed Student's t -test). Blots in A and D are representative of three repeats. Inputs are 7.5%.

Article Snippet: The following primary antibodies were used at 1:200: anti-survivin (Cell Signaling Technologies 71G4B7 or 6E4), anti-H3K27Me3 (Abcam, Ab192985 ), anti-EZH2 [Cell Signalling Technologies (CST), D269].

Techniques: Immunoprecipitation, Negative Control, GST Pulldown Assay, Expressing, Western Blot, Two Tailed Test

Journal: bioRxiv

Article Title: SUN2 mediates epigenetic remodeling to drive mechanotransduction during skin fibrosis

doi: 10.64898/2026.03.19.712957

Figure Lengend Snippet: (A-C) Images and quantification of Ezh2 immunofluorescence in WT and Sun2 KO dermal fibroblasts cultured on soft or stiff substrates. (A) Scale bar = 10 μ m. (B) Quantification of nuclear Ezh2 intensity. N = 2, n soft = 21–37, n stiff = 20–24. Significance was assessed using a two-tailed Mann–Whitney U test. (C) qPCR analysis of Ezh2 mRNA expression in WT or Sun2 KO fibroblasts on soft or stiff substrates. N = 3. Significance was assessed using a two-tailed Mann–Whitney U test. (D-E) Schematic and analysis of WT and Sun2 KO dermal fibroblasts plated on soft or stiff substrates for 24 h, followed by 24 h treatment with the Ezh2 inhibitor GSK343 or DMSO (Day 1), and collection for qPCR (Day 2). N = 3 independent experiments. Statistical significance was assessed using one-way ANOVA. ns, not significant; p < 0.05, * p < 0.01, ** p < 0.001, *** p < 0.0001. (F) Proposed model for SUN2–LINC–PRC2–dependent control of profibrotic gene expression in dermal fibroblasts. In quiescent WT fibroblasts, fibrotic genes are repressed. Upon mechanical stimulation of WT cells, cytoskeletal tension transmitted through LINC complexes promotes lamin stretching and altered Ezh2-driven epigenetic remodeling to activate transcription, repress transcriptional activators, and promote enhancer-licensing. In Sun2 KO dermal fibroblasts,loss of LINC complexes blunts this mechanotransduction despite cytoskeletal activation.

Article Snippet: Primary antibody diluted in blocking solution – SUN2, 1:200 (Abcam, AB124916), YAP, 1:200 (CST, 4912S), LaminA/C (Abcam, AB133256), and/or Ezh2, 1:200 (CST, 5246T) was incubated overnight at 4°C.

Techniques: Immunofluorescence, Cell Culture, Two Tailed Test, MANN-WHITNEY, Expressing, Control, Gene Expression, Activation Assay

Journal: The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology

Article Title: LncRNA KCNQ1OT1 aggravates myocardial ischemia/reperfusion injury by mediating the EZH2/TIMP-3 axis

doi: 10.4196/kjpp.25.148

Figure Lengend Snippet: (A) EZH2 enrichment by KCNQ1OT1 was tested by RIP assay. (B) The binding relationship of KCNQ1OT1 and EZH2 was assessed by RNA pull-down assay. (C) EZH2 and H3K27me3 enrichment in the promoter region of TIMP-3 was measured by ChIP assay. (D) TIMP-3 expression levels after sh-KCNQ1OT1 treatment were assessed by RT-qPCR. (E, F) The protein expression levels of EZH2 and TIMP-3 were assessed by Western blot. (G) The localization relationship between KCNQ1OT1 and EZH2 in the nucleus was measured using FISH combined with IF. Scale bar = 10 μm. Data are presented as mean ± standard deviation. For multiple-group analyses, one-way ANOVA was used, followed by Tukey’s post-hoc test for pairwise comparisons. *p < 0.05; N = 3. KCNQ1OT1, potassium voltage-gated channel subfamily q member 1 overlapping transcript 1; EZH2, enhancer of zeste homolog 2; TIMP-3, tissue inhibitor of metalloproteinase-3; RIP, RNA immunoprecipitation; ChIP, chromatin immunoprecipitation; RT-qPCR, reverse transcription quantitative polymerase chain reaction; FISH, fluorescence in situ hybridization; IF, immunofluorescence; IgG, immunoglobulin G; DMSO, dimethyl sulfoxide.

Article Snippet: After centrifugation, the concentration of protein was determined by the bicinchoninic acid method (Beyotime), and then treated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis for 2 h, transferred to a membrane and sealed by 5% skim milk powder for 2 h. The membrane was added with primary antibodies EZH2 (1:1,000, Cell Signaling Technology), TIMP-3 (1:1,000, Sigma-Aldrich) and GAPDH (1:1,000, Cell Signaling Technology) 4 ml at 4°C overnight, then incubated for 1 h with 4 ml secondary antibody immunoglobulin G (IgG)/horseradish peroxidase.

Techniques: Binding Assay, Pull Down Assay, Expressing, Quantitative RT-PCR, Western Blot, Standard Deviation, RNA Immunoprecipitation, Chromatin Immunoprecipitation, Reverse Transcription, Real-time Polymerase Chain Reaction, Fluorescence, In Situ Hybridization, Immunofluorescence

Journal: The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology

Article Title: LncRNA KCNQ1OT1 aggravates myocardial ischemia/reperfusion injury by mediating the EZH2/TIMP-3 axis

doi: 10.4196/kjpp.25.148

Figure Lengend Snippet: (A) EZH2 enrichment by KCNQ1OT1 was tested by RIP assay. (B) The binding relationship of KCNQ1OT1 and EZH2 was assessed by RNA pull-down assay. (C) EZH2 and H3K27me3 enrichment in the promoter region of TIMP-3 was measured by ChIP assay. (D) TIMP-3 expression levels after sh-KCNQ1OT1 treatment were assessed by RT-qPCR. (E, F) The protein expression levels of EZH2 and TIMP-3 were assessed by Western blot. (G) The localization relationship between KCNQ1OT1 and EZH2 in the nucleus was measured using FISH combined with IF. Scale bar = 10 μm. Data are presented as mean ± standard deviation. For multiple-group analyses, one-way ANOVA was used, followed by Tukey’s post-hoc test for pairwise comparisons. *p < 0.05; N = 3. KCNQ1OT1, potassium voltage-gated channel subfamily q member 1 overlapping transcript 1; EZH2, enhancer of zeste homolog 2; TIMP-3, tissue inhibitor of metalloproteinase-3; RIP, RNA immunoprecipitation; ChIP, chromatin immunoprecipitation; RT-qPCR, reverse transcription quantitative polymerase chain reaction; FISH, fluorescence in situ hybridization; IF, immunofluorescence; IgG, immunoglobulin G; DMSO, dimethyl sulfoxide.

Article Snippet: After washing, the cells were blocked with blocking solution (5% BSA) at room temperature for 1 h, and then incubated with an anti-EZH2 primary antibody (Cell Signaling Technology, Cat#5246, diluted at 1:200) at 4°C overnight.

Techniques: Binding Assay, Pull Down Assay, Expressing, Quantitative RT-PCR, Western Blot, Standard Deviation, RNA Immunoprecipitation, Chromatin Immunoprecipitation, Reverse Transcription, Real-time Polymerase Chain Reaction, Fluorescence, In Situ Hybridization, Immunofluorescence