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 is nuclear in hypoxic cells. (A) U2OS cells were subjected to hypoxia (1% O 2 ) for 24 h hypoxia then fixed and stained to highlight endogenous survivin (green). Nuclei were stained with NucBlue (blue). Scale bar: 50 µm. (B) Graph illustrating colocalisation analysis using Pearson's correlation coefficient score, based on the signals in A. The results are expressed as mean±s.d. from three independent experiments with n =250 cells. **** P <0.0001 (unpaired two-tailed Student's t -test). (C,D) Immunoblot analysis of U2OS cell extracts subjected to fractionation. Nuclear and cytoplasmic fractions are indicated by positive anti-TBP and tubulin bands, respectively. Survivin was more abundant in the nuclear fraction under hypoxic conditions. (E) Quantification of percentage of nuclear survivin in normoxic versus hypoxic samples using total survivin protein. Results are mean±s.d., n =3. ** P =0.0025 (unpaired two-tailed Student's t -test). (F) Time-point analysis of U2OS cells expressing survivin–GFP (green) upon exposure to hypoxia. Cells were fixed and stained with NucBlue (blue) then imaged. Scale bars: 50 µm. (G) Colocalisation analysis of images in F revealed significant differences in Pearson's correlation coefficient scores when cells were subjected to hypoxia at all time intervals as compared to 0 h, with maximum nuclear expression achieved in 3 h. Results are mean±s.d., n =300 cells **** P <0.0001 (one-way ANOVA followed by Dunnett's post hoc test post test). (H) U2OS cells expressing survivin–GFP that had been exposed to hypoxia for 24 h were returned to normoxia for 3 or 6 h. Over this time frame survivin was retained in the nucleus. Control is normoxia. Scale bars: 50 µm. (I) Quantification of data shown in H. Results are mean±s.d., n =100 cells in each of three biological repeats. **** P <0.0001 (one-way ANOVA with Dunnett's post hoc test).

Article Snippet: The siRNA survivin (s1457 and s1458 from Thermo Fisher Scientific), SignalSilence ® Survivin siRNA II (Cell Signalling Technology) and siRNA control (Thermo Fisher Scientific) were diluted in medium without antibiotics and supplemented with RNAiMAX transfection reagent for 10 min.

Techniques: Staining, Two Tailed Test, Western Blot, Fractionation, Expressing, 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: 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 siRNA survivin (s1457 and s1458 from Thermo Fisher Scientific), SignalSilence ® Survivin siRNA II (Cell Signalling Technology) and siRNA control (Thermo Fisher Scientific) were diluted in medium without antibiotics and supplemented with RNAiMAX transfection reagent for 10 min.

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 siRNA survivin (s1457 and s1458 from Thermo Fisher Scientific), SignalSilence ® Survivin siRNA II (Cell Signalling Technology) and siRNA control (Thermo Fisher Scientific) were diluted in medium without antibiotics and supplemented with RNAiMAX transfection reagent for 10 min.

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: The siRNA survivin (s1457 and s1458 from Thermo Fisher Scientific), SignalSilence ® Survivin siRNA II (Cell Signalling Technology) and siRNA control (Thermo Fisher Scientific) were diluted in medium without antibiotics and supplemented with RNAiMAX transfection reagent for 10 min.

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 knockdown reduces transcription of several key PRC2-dependent genes. qPCR analysis was carried out for the genes indicated from (A) U2OS or (B) MRC5 cells treated with either control or survivin-specific siRNA (48 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; **** P <0.0001; ns, not significant (two-way ANOVA with Tukey's multiple comparisons post test). (C) qPCR analysis of major satellite transcripts from U2OS or MRC5 cells exposed to control or survivin-specific siRNA. A significant reduction in major satellite expression occurred in the absence of survivin in both lines. Data are normalized to control siRNA treatment and means±s.d. from n =3 plotted. * P <0.05 (two-way ANOVA with Tukey's multiple comparisons post test).

Article Snippet: The siRNA survivin (s1457 and s1458 from Thermo Fisher Scientific), SignalSilence ® Survivin siRNA II (Cell Signalling Technology) and siRNA control (Thermo Fisher Scientific) were diluted in medium without antibiotics and supplemented with RNAiMAX transfection reagent for 10 min.

Techniques: Knockdown, Control, 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: The siRNA survivin (s1457 and s1458 from Thermo Fisher Scientific), SignalSilence ® Survivin siRNA II (Cell Signalling Technology) and siRNA control (Thermo Fisher Scientific) were diluted in medium without antibiotics and supplemented with RNAiMAX transfection reagent for 10 min.

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: Model of survivin–PRC2 interaction and its impact on transcription. Survivin inhibits the PRC2 complex causing a reduction in H3K27 tri-methylation, which opens the DNA to enable transcription of genes that are normally repressed by PRC2.

Article Snippet: The siRNA survivin (s1457 and s1458 from Thermo Fisher Scientific), SignalSilence ® Survivin siRNA II (Cell Signalling Technology) and siRNA control (Thermo Fisher Scientific) were diluted in medium without antibiotics and supplemented with RNAiMAX transfection reagent for 10 min.

Techniques: Methylation

Journal: Tumour Virus Research

Article Title: Ad-VT oncolytic adenovirus suppresses bladder cancer via cAMP-dependent AMPK-Raptor activation and G2/M arrest

doi: 10.1016/j.tvr.2026.200337

Figure Lengend Snippet: Ad-VT activates AMPK to block cell cycle progression. (A, B) The cell cycle distribution of UM-UC-3 cells were detected by flow cytometry. UM-UC-3 cells were first treated with Dorsomorphina (1.25 μM) or transfected with siRNA, and then infected with the viruses for 48 h (MOI = 100). (C – E) Western blot detection of the expression levels of cell cycle related proteins showing the inhibition of the AMPK pathway, following infection for 48 h (MOI = 100). (F) Western blot detection of the expression levels of cell cycle related proteins showing the activation of the AMPK pathway, following infection for 48 h (MOI = 100). Data are presented as mean ± SEM, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: Raptor siRNA (sc-44067) and AMPK siRNA (siG000005562 A) were designed and synthesized by Santa Cruz Biotechnology, Inc.

Techniques: Blocking Assay, Flow Cytometry, Transfection, Infection, Western Blot, Expressing, Inhibition, Activation Assay

Journal: Tumour Virus Research

Article Title: Ad-VT oncolytic adenovirus suppresses bladder cancer via cAMP-dependent AMPK-Raptor activation and G2/M arrest

doi: 10.1016/j.tvr.2026.200337

Figure Lengend Snippet: Inhibitory effect of Ad-VT on UM-UC-3 cells in vitro and in vivo through the AMPK-Raptor pathway. (A, B) The expression levels of p-AMPK, AMPK, p-Raptor, Raptor, p-mTOR, and mTOR at 24h after Ad-VT infection (MOI = 100) of UM-UC-3 cells and pretreatment (2h) with a vehicle, Dorsomorphin (1.25 μM) or transfection with the control or Raptor siRNA. (C, D) The viability of UM-UC-3 cells and 5637 cells that were pretreated with Dorsomorphin (B) or Raptor siRNA (C). (E, F) Effect of Dorsomorphin on tumor volume (D) and survival rate (E) of nude mice treated with PBS or Ad-VT. (G) The effect of Raptor siRNA on survival rate of nude mice treated with PBS or Ad-VT treatment. Data are presented as mean ± SEM, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: Raptor siRNA (sc-44067) and AMPK siRNA (siG000005562 A) were designed and synthesized by Santa Cruz Biotechnology, Inc.

Techniques: In Vitro, In Vivo, Expressing, Infection, Transfection, Control

Journal: Journal of Translational Autoimmunity

Article Title: A novel epigenetic regulation of JAM-A by EZH2-DNMT3A cascade contributes to T cell adhesion via the activation of Rap1a in lupus patients

doi: 10.1016/j.jtauto.2026.100362

Figure Lengend Snippet: Increased expression of JAM-A in SLE patient PBMCs and peripheral T cells. (A – D) FCM analysis of JAM-A + PBMCs and JAM-A + T cells. (A) JAM-A was stained with anti-human JAM-A-PE antibodies in the PBMCs of healthy individuals (red), new SLE patients (blue), SLE treated (green) and isotype controls (black). (B) Quantification of JAM-A + PBMCs from (A). (C) Double staining with both anti-human JAM-A-PE and anti-human CD3-FITC antibodies. (D) Quantification of JAM-A + T cells from (C), and the error bars indicate the SEM. Healthy, (healthy volunteers, n = 24); new SLE (SLE patients without clinical treatments, n = 9); SLE treated (SLE patients with clinical treatments, n = 15). (E – F) The correlations of EZH2 mRNA/β-actin and the JAM-A mRNA/β-actin in PBMCs ( E ) and T cells ( F ) from SLE patients. ∗ P < 0.05, ∗∗∗ P < 0.001.

Article Snippet: Cells were stimulated overnight with anti-CD3 and anti-CD28 antibodies and transfected with 100 nM EZH2 siRNAs (siG09121182926-1-5/si-h-EZH2_001 and siG09121182954-1-5/si-h-EZH2_002, RiboBio, China), JAM-A siRNAs (SC-43139, Santa Cruz Biotechnology, USA), and negative control siRNA (siN0000001-1-5 for EZH2, SC-37007 for JAM-A) in 6-well plates.

Techniques: Expressing, Staining, Double Staining

Journal: Journal of Translational Autoimmunity

Article Title: A novel epigenetic regulation of JAM-A by EZH2-DNMT3A cascade contributes to T cell adhesion via the activation of Rap1a in lupus patients

doi: 10.1016/j.jtauto.2026.100362

Figure Lengend Snippet: The crosstalk between EZH2/H3K27me3 and DNMT3A/JAM-A. (A, D) The protein expression in Jurkat cells or T cells treated with 5 μM GSK126 (A) or EZH2-siRNA (D), GAPDH or H3 protein as loading control. (B, E) The methylation frequency in JAM-A gene promoter of T cells under EZH2 inhibitor 5 μM GSK126 (B) or EZH2-siRNA treated (E). (C, F) Immunoprecipitated JAM-A with anti-DNMT3A or DNMT3A with anti-H3K27me3 ChIP-grade antibodies in T cells under 5 μM GSK126 (C) or EZH2-siRNA treated (F). The values were normalized to the chromatin levels with 1% input samples. (G, H) The negative correlations of EZH2 mRNA/β-actin and DNMT3A mRNA/β-actin (G) , DNMT3A mRNA/β-actin and JAM-A mRNA/β-actin (H) in T cells from SLE patients. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, and error bars indicate the SEM.

Article Snippet: Cells were stimulated overnight with anti-CD3 and anti-CD28 antibodies and transfected with 100 nM EZH2 siRNAs (siG09121182926-1-5/si-h-EZH2_001 and siG09121182954-1-5/si-h-EZH2_002, RiboBio, China), JAM-A siRNAs (SC-43139, Santa Cruz Biotechnology, USA), and negative control siRNA (siN0000001-1-5 for EZH2, SC-37007 for JAM-A) in 6-well plates.

Techniques: Expressing, Control, Methylation, Immunoprecipitation

Journal: Journal of Translational Autoimmunity

Article Title: A novel epigenetic regulation of JAM-A by EZH2-DNMT3A cascade contributes to T cell adhesion via the activation of Rap1a in lupus patients

doi: 10.1016/j.jtauto.2026.100362

Figure Lengend Snippet: Inhibition of EZH2 and JAM-A suppresses β1 integrin-mediated T cells and Jurkat cells adhering to ECM. ( A ) The adhesion capacity in T cells from Healthy and SLE patients in vitro . ( B-C ) The adhesion capacity of T cells isolated from healthy or Jurkat cells blocked by 5 μM GSK126 (B) or EZH2-siRNA (C) in vitro . ( D ) Compared the adhesion capacity of T cells from SLE patients, or Jurkat cells with/out neutralizing anti-JAM-A, 1 μg/ml J10.4. (E) Protein expression levels in Jurkat cells overexpressing or knockdown of JAM-A. (F) The adhesion capacity of Jurkat cells with JAM-A regulation. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗ P < 0.001, and error bars indicate the SEM.

Article Snippet: Cells were stimulated overnight with anti-CD3 and anti-CD28 antibodies and transfected with 100 nM EZH2 siRNAs (siG09121182926-1-5/si-h-EZH2_001 and siG09121182954-1-5/si-h-EZH2_002, RiboBio, China), JAM-A siRNAs (SC-43139, Santa Cruz Biotechnology, USA), and negative control siRNA (siN0000001-1-5 for EZH2, SC-37007 for JAM-A) in 6-well plates.

Techniques: Inhibition, In Vitro, Isolation, Expressing, Knockdown

Journal: Journal of Translational Autoimmunity

Article Title: A novel epigenetic regulation of JAM-A by EZH2-DNMT3A cascade contributes to T cell adhesion via the activation of Rap1a in lupus patients

doi: 10.1016/j.jtauto.2026.100362

Figure Lengend Snippet: Inhibition of EZH2 with GSK126 ameliorates lupus-like disease phenotypes in MRL/ lpr mice. (A) Eight-week-old female MRL/ lpr mice were adaptively fed for 2 weeks (wks, n = 15) in the SPF animal room. Mice were randomly divided into a vehicle group (n = 8) and a GSK126-treated group (n = 7) and were treated with an intraperitoneal (IP) injection of β-SEB (20%) or GSK126 (50 mg/kg) every other day for one month. (B) Survival curves of the vehicle and treated group. (C) Anti-dsDNA antibody levels in the plasma. (D) The mRNA expression of IL-10 and TGF-β in splenomegaly. (E) Photomicrographic representation of renal damage (arrow). (F) The percentage plot of glomerulus damage. (G) The Representative histological images of immunohistochemical staining of JAM-A and β1-integrin antibody. (H) The quantification of positive staining areas was quantified by Saiviewer software. (I) The protein expression of EZH2, DNMT3A, JAM-A, Rap1a, β1 integrin and H3K27me3 in splenocytes detected by western blotting. (J) A total of 7 mice per group were analyzed for densitometry by image J software from (I). The symbols represent individual mice, and error bars indicate the SEM. ∗P < 0.05, ∗∗∗P < 0.001, ∗∗∗P < 0.001.

Article Snippet: Cells were stimulated overnight with anti-CD3 and anti-CD28 antibodies and transfected with 100 nM EZH2 siRNAs (siG09121182926-1-5/si-h-EZH2_001 and siG09121182954-1-5/si-h-EZH2_002, RiboBio, China), JAM-A siRNAs (SC-43139, Santa Cruz Biotechnology, USA), and negative control siRNA (siN0000001-1-5 for EZH2, SC-37007 for JAM-A) in 6-well plates.

Techniques: Inhibition, Injection, Clinical Proteomics, Expressing, Immunohistochemical staining, Staining, Software, Western Blot

Journal: Journal of Translational Autoimmunity

Article Title: A novel epigenetic regulation of JAM-A by EZH2-DNMT3A cascade contributes to T cell adhesion via the activation of Rap1a in lupus patients

doi: 10.1016/j.jtauto.2026.100362

Figure Lengend Snippet: Proposed schematic model of the EZH2/miR-26a-5p signaling axis involved in the development of SLE. Increased EZH2 expression is mediated by miR-26a-5p, which in turn is epigenetically repressed by EZH2-mediated H3K27me3 trimethylation within the miR-26a-5p promoter, thus forming a vicious cycle. Crosstalk between H3K27me3 and CpG island methylation mediated by DNMT3A within the JAM-A promoter, resulted in increased expression of JAM-A. Increased expression of JAM-A up-regulated the expression of Rap1a, a regulator of β1-integrin, which is functionally relevant to T cell adhesion. Combined with the increased transcriptional level of Rap1a mediated by miR-26a-5p, ultimately, Rap1a led to increased expression of β1-integrin, which is involved in increasing T cell adhesion.

Article Snippet: Cells were stimulated overnight with anti-CD3 and anti-CD28 antibodies and transfected with 100 nM EZH2 siRNAs (siG09121182926-1-5/si-h-EZH2_001 and siG09121182954-1-5/si-h-EZH2_002, RiboBio, China), JAM-A siRNAs (SC-43139, Santa Cruz Biotechnology, USA), and negative control siRNA (siN0000001-1-5 for EZH2, SC-37007 for JAM-A) in 6-well plates.

Techniques: Expressing, Methylation