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: Nature Communications

Article Title: HnRNP A1 and A2B1 enforce Ezh2 mRNA splicing to promote germinal center B cell responses

doi: 10.1038/s41467-026-74159-w

Figure Lengend Snippet: A Changes in five types of alternative splicing events upon hnRNP A1/A2B1 knockout. Scatter plots show exon inclusion level differences between Hnrnpa1 f/f Hnrnpa2b1 f/f (WT) and Hnrnpa1 f/f Hnrnpa2b1 f/f Cd19 Cre/+ (double knockout, DKO) naïve B cells. Each dot represents a significantly altered splicing event (|inclusion level difference| > 20%, P < 0.05). B Lollipop plot showing the top 10 most significant exon-skipping events (ranked by P -value) in naïve B cells from DKO mice. The height of each lollipop indicates the inclusion level difference, and the circle size reflects statistical significance. C RNA expression changes of the top 10 most significant exon-skipping target genes (ranked by P -value) in follicular and Germinal center (GC) B cells from WT mice. Circle size and color intensity represent changes in FPKM values. Data from GEO: GSE15907 . D–H Alternative splicing of Ezh2 following hnRNP A1/A2B1 deletion. D Sashimi plot illustrating exon 14 splicing changes in Ezh2 in WT and DKO naïve B cells, showing both the full-length isoform and the exon 14-skipped isoform ( Ezh2Δ14 ). E Quantification of splice junction reads spanning exons 13–14, 14–15, and 13–15 in WT and DKO naïve B cells ( n = 3), data in ( E ) are from a single experiment (RNA-seq). F Schematic and primer design for detecting full-length ( Ezh2-FL ) and exon 14-skipped ( Ezh2Δ14 ) isoforms. G Semi-quantitative PCR showing the effect of single and double hnRNP knockouts on Ezh2 exon 14 skipping in WT and DKO naïve B cells, with accompanying gel image and the ratio of Ezh2Δ14 to Ezh2-FL . The data shown are representative images from two independent experiments. H Quantification of Ezh2-FL and Ezh2Δ14 expression normalized to β-actin in WT and DKO naïve B cells ( n = 2), the data shown here represent one representative set of statistical results from two independent experiments. I , J Western blot analysis detecting the abundance of H3K27me3 in B cells from WT, A1-SKO, A2B1-SKO, and DKO mice after 48 h of stimulation with anti-CD40 (1 μg/mL) and anti-IgM (2 μg/mL). The data shown are representative images from two independent experiments. K Immunoblot analysis of H3K27me3 levels in WT and DKO B cells. Cells were pre-stimulated with anti-CD40 and anti-IgM and subsequently reconstituted with an empty vector (Vector), full-length EZH2 ( Ezh2 -FL), or the exon 14–skipped isoform ( Ezh2 Δ14). The data shown are representative images from two independent experiments. Each symbol represents an individual mouse ( E , H ). All data are shown as mean ± SEM. Results are representative of at least two independent experiments ( H ). Statistical significance was assessed using an unpaired two-tailed Student’s t -test ( E ).

Article Snippet: Primary antibodies used were: β-actin (1:5000, Proteintech, 20536-1-AP), hnRNP A1 (1:1000, Santa Cruz Biotechnology, sc-32301), hnRNP A2B1 (1:1000, Santa Cruz Biotechnology, sc-53531), EZH2 (1:1000, ABclonal, A5743; A19577), H3K27me3 (1:1000, ABclonal, A22396), CDKN1A (1:1000, Cell Signaling Technology, 2947).

Techniques: Alternative Splicing, Knock-Out, Double Knockout, RNA Expression, RNA Sequencing, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Plasmid Preparation, Two Tailed Test

Journal: Nature Communications

Article Title: HnRNP A1 and A2B1 enforce Ezh2 mRNA splicing to promote germinal center B cell responses

doi: 10.1038/s41467-026-74159-w

Figure Lengend Snippet: A Volcano plot showing differentially expressed genes identified by RNA-seq in Hnrnpa1 f/f Hnrnpa2b1 f/f (WT) and Hnrnpa1 f/f Hnrnpa2b1 f/f Cd19 Cre/+ (double knockout, DKO) B cells stimulated with anti-CD40 (1 μg/mL) and anti-IgM (2 μg/mL) for 48 h. Differentially expressed genes are defined as those with |log 2 FC| > 1 and p < 0.05. CDKN1A is significantly upregulated in DKO B cells. Differential gene expression analysis for RNA-seq data was performed using DESeq2, which models count data based on the negative binomial distribution. Statistical significance was assessed using two-sided tests. P -values were adjusted for multiple comparisons using the Benjamini–Hochberg method to control the false discovery rate (FDR). Genes with an adjusted P value (FDR) < 0.05 were considered significantly differentially expressed. B qRT-PCR analysis of CDKN1A expression in naïve B cells, B cells stimulated with anti-CD40 (1 μg/mL) and anti-IgM (2 μg/mL) for 48 h, and early Germinal center (GC) B cells isolated 5 days after NP-KLH/Alum immunization (naïve B : n = 6; anti-CD40 and anti-IgM: n = 6; early-GC: n = 5). C , D Western blot analysis of CDKN1A protein expression in unstimulated and 48-h-stimulated B cells ( n = 3). The data shown are represe n tative images from three independent experiments. E , F ChIP-qPCR analysis of Cdkn1a locus enriched by anti-H3K27me3 antibody in B cells from WT and DKO mice after 48 h of stimulation with anti-CD40 (1 μg/mL) and anti-IgM (2 μg/mL). E A schematic representation of the Cdkn1a gene showing exons and the putative H3K27me3-enriched regions, P1 and P2. F ChIP-qPCR analysis validated the enrichment of H3K27me3 at the Cdkn1a genomic loci in WT and DKO B cells. Data are presented as fold enrichment relative to the WT IgG control. p < 0.0001( p = 2.06e-6). G , H CFSE dilution assay assessing proliferation of WT and DKO B cells following 72 h of anti-CD40 (1 μg/mL) and anti-IgM (2 μg/mL) stimulation ( n = 4). G Representative flow cytometry histograms showing CFSE dilution. H Quantitative analysis of CFSE dilution reflecting proliferation capacity ( n = 4; two independent experiments). Each symbol represents an individual mouse ( B , D , F , H ). Data are shown as mean ± SEM. Results shown are representative of at least three independent experiments ( B , D , F , H ). Statistical significance was determined using an unpaired two-tailed Student’s t -test ( B , D , H ) or one-way ANOVA followed by Tukey’s multiple comparisons test ( F ).

Article Snippet: Primary antibodies used were: β-actin (1:5000, Proteintech, 20536-1-AP), hnRNP A1 (1:1000, Santa Cruz Biotechnology, sc-32301), hnRNP A2B1 (1:1000, Santa Cruz Biotechnology, sc-53531), EZH2 (1:1000, ABclonal, A5743; A19577), H3K27me3 (1:1000, ABclonal, A22396), CDKN1A (1:1000, Cell Signaling Technology, 2947).

Techniques: RNA Sequencing, Double Knockout, Gene Expression, Control, Quantitative RT-PCR, Expressing, Isolation, Western Blot, ChIP-qPCR, Dilution Assay, Flow Cytometry, Two Tailed Test

Journal: Nature Communications

Article Title: HnRNP A1 and A2B1 enforce Ezh2 mRNA splicing to promote germinal center B cell responses

doi: 10.1038/s41467-026-74159-w

Figure Lengend Snippet: A Changes in five types of alternative splicing events upon hnRNP A1/A2B1 knockout. Scatter plots show exon inclusion level differences between Hnrnpa1 f/f Hnrnpa2b1 f/f (WT) and Hnrnpa1 f/f Hnrnpa2b1 f/f Cd19 Cre/+ (double knockout, DKO) naïve B cells. Each dot represents a significantly altered splicing event (|inclusion level difference| > 20%, P < 0.05). B Lollipop plot showing the top 10 most significant exon-skipping events (ranked by P -value) in naïve B cells from DKO mice. The height of each lollipop indicates the inclusion level difference, and the circle size reflects statistical significance. C RNA expression changes of the top 10 most significant exon-skipping target genes (ranked by P -value) in follicular and Germinal center (GC) B cells from WT mice. Circle size and color intensity represent changes in FPKM values. Data from GEO: GSE15907 . D–H Alternative splicing of Ezh2 following hnRNP A1/A2B1 deletion. D Sashimi plot illustrating exon 14 splicing changes in Ezh2 in WT and DKO naïve B cells, showing both the full-length isoform and the exon 14-skipped isoform ( Ezh2Δ14 ). E Quantification of splice junction reads spanning exons 13–14, 14–15, and 13–15 in WT and DKO naïve B cells ( n = 3), data in ( E ) are from a single experiment (RNA-seq). F Schematic and primer design for detecting full-length ( Ezh2-FL ) and exon 14-skipped ( Ezh2Δ14 ) isoforms. G Semi-quantitative PCR showing the effect of single and double hnRNP knockouts on Ezh2 exon 14 skipping in WT and DKO naïve B cells, with accompanying gel image and the ratio of Ezh2Δ14 to Ezh2-FL . The data shown are representative images from two independent experiments. H Quantification of Ezh2-FL and Ezh2Δ14 expression normalized to β-actin in WT and DKO naïve B cells ( n = 2), the data shown here represent one representative set of statistical results from two independent experiments. I , J Western blot analysis detecting the abundance of H3K27me3 in B cells from WT, A1-SKO, A2B1-SKO, and DKO mice after 48 h of stimulation with anti-CD40 (1 μg/mL) and anti-IgM (2 μg/mL). The data shown are representative images from two independent experiments. K Immunoblot analysis of H3K27me3 levels in WT and DKO B cells. Cells were pre-stimulated with anti-CD40 and anti-IgM and subsequently reconstituted with an empty vector (Vector), full-length EZH2 ( Ezh2 -FL), or the exon 14–skipped isoform ( Ezh2 Δ14). The data shown are representative images from two independent experiments. Each symbol represents an individual mouse ( E , H ). All data are shown as mean ± SEM. Results are representative of at least two independent experiments ( H ). Statistical significance was assessed using an unpaired two-tailed Student’s t -test ( E ).

Article Snippet: Primary antibodies used were: β-actin (1:5000, Proteintech, 20536-1-AP), hnRNP A1 (1:1000, Santa Cruz Biotechnology, sc-32301), hnRNP A2B1 (1:1000, Santa Cruz Biotechnology, sc-53531), EZH2 (1:1000, ABclonal, A5743; A19577), H3K27me3 (1:1000, ABclonal, A22396), CDKN1A (1:1000, Cell Signaling Technology, 2947).

Techniques: Alternative Splicing, Knock-Out, Double Knockout, RNA Expression, RNA Sequencing, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Plasmid Preparation, Two Tailed Test

Journal: Nature Communications

Article Title: HnRNP A1 and A2B1 enforce Ezh2 mRNA splicing to promote germinal center B cell responses

doi: 10.1038/s41467-026-74159-w

Figure Lengend Snippet: A RIP-qPCR analysis showing enrichment of Ezh2 transcripts associated with hnRNP A1 or hnRNP A2B1 in splenic B cells from WT mice, with B cells from Hnrnpa1 f/f Hnrnpa2b1 f/f Cd19 Cre/+ (double knockout, DKO) mice used as a control for RIP ( n = 2). Data are shown as mean ± SD from a single experiment. Statistical analysis was performed using an unpaired two-tailed Student’s t -test. B RBPmap prediction of hnRNP A1 and hnRNP A2B1 binding sites in Ezh2 . The analysis used exon 14 and part of intron 14 of Ezh2 as input. Underlined regions denote potential shared binding sites for hnRNP A1 and A2B1. C–E Construction and analysis of an Ezh2 minigene to assess exon 14 skipping upon site-directed mutation of predicted binding sites. C Schematic of the Ezh2 minigene and the design of mutations at predicted binding sites (M1, M2, and M3). Additional constructs include double (M1 + M3) and triple (M1 + M2 + M3) mutants. D , E WT and DKO B cells were infected with retroviruses carrying minigene constructs (WT, M1, M3, M1 + M3, and M1 + M2 + M3). 48 h after infection, GFP⁺ B cells were sorted by flow cytometry and subjected to RT-PCR analysis of Ezh2 splicing isoforms. The gel image shows the relative abundance of Ezh2Δ14 and Ezh2-FL isoforms. The data shown are representative images from two independent experiments. F–I RNA pull-down assay to evaluate binding of wild-type and mutant Ezh2 pre-RNAs to hnRNP A1 and A2B1. F Schematic of the RNA pull-down workflow. B cells were purified using CD19 magnetic beads, nuclear extracts were prepared, and biotin-labeled RNAs were pulled down using Streptavidin Magnetic Beads. G Biotin-labeled RNAs synthesized for pull-down: wild-type intron 14 RNA (I14–WT–Biotin), reverse complementary RNA (I14–Antisense–Biotin), triple mutant (I14–M1 + M2 + M3–Biotin), and M1-only mutant (I14–M1–Biotin). H Immunoblot analysis of proteins pulled down with biotin-labeled RNAs (I14–M1 + M2 + M3–Biotin), detecting hnRNP A1 and A2B1. I RNA pull-down assay was performed using the biotin-labeled RNA (I14–M1–Biotin), followed by immunoblotting to detect the precipitated hnRNP A1 and hnRNP A2B1 proteins. The data shown are representative images from two independent experiments. J–M RNA EMSA to determine direct binding of hnRNP A1 and A2B1 to wild-type or mutant Ezh2 pre-mRNA. J EMSA with hnRNP A1 and wild-type vs. M1 + M2 + M3 mutant Ezh2 pre-RNA. K EMSA with hnRNP A2B1 and wild-type vs. M1 + M2 + M3 mutant Ezh2 pre-RNA. L EMSA with hnRNP A1 and wild-type vs. M1 mutant Ezh2 pre-RNA. M EMSA with hnRNP A2B1 and wild-type vs. M1 mutant Ezh2 pre-RNA. The data shown are representative images from two independent experiments. All data are shown as mean ± SEM. Results shown are representative of three independent experiments ( A ). Statistical significance was assessed using an unpaired two-tailed Student’s t -test ( A ).

Article Snippet: Primary antibodies used were: β-actin (1:5000, Proteintech, 20536-1-AP), hnRNP A1 (1:1000, Santa Cruz Biotechnology, sc-32301), hnRNP A2B1 (1:1000, Santa Cruz Biotechnology, sc-53531), EZH2 (1:1000, ABclonal, A5743; A19577), H3K27me3 (1:1000, ABclonal, A22396), CDKN1A (1:1000, Cell Signaling Technology, 2947).

Techniques: Double Knockout, Control, Two Tailed Test, Binding Assay, Mutagenesis, Construct, Infection, Flow Cytometry, Reverse Transcription Polymerase Chain Reaction, Pull Down Assay, Purification, Magnetic Beads, Labeling, Synthesized, Western Blot

Journal: Neoplasia (New York, N.Y.)

Article Title: BPGM as an intrinsic brake to constrain metastasis through phospho-epigenetic-mediated carnitine biosynthesis suppression

doi: 10.1016/j.neo.2026.101299

Figure Lengend Snippet: 2,3-BPG-CDK1-EZH2-H3K27me3 Axis: BPGM’s epigenetic circuit breaker for cellular migration. (A) Integrated functional metabolomics analysis revealed BPGM-altered metabolites clustered in methyl donor group. Bubble size: metabolites count. (B) Hypothesis of molecular mechanism underlying BPGM regulated BBOX1 expression by post transcriptional modification (PTM). (C) Silencing BPGM significantly reduced the protein level of H3K27me3, while overexpressing BPGM increased its level. Cells stably expressing shBPGM/BPGM and its control cells (shCtrl/Ctrl) were used to detect protein level by western blotting. (D) ChIP assays disclosed that the fragments of BBOX1 and MMP9 promoter precipitated by anti-H3K27me3 antibody were increased upon overexpressing BPGM. SK-HEP-1 cells stably expressing BPGM and its control cells (Ctrl) were employed to ChIP assay. The antibody precipitated DNAs were amplified by qPCR. 5 % of the total DNAs were amplified to serve as the control for DNA content. Values shown are signal of α-H3K27me3-precipitated DNA relative to the input and the mean value of the control group was normalized as 1. (E) Overexpressing BPGM significantly increased the protein level of EZH2 but decreased the protein level of p-EZH2-T 345 in tumor cells. (F) The molecular docking of 2,3-BPG and CDK1. Predicted structure of 2,3-BPG binding with CDK1. Key contact residues: Thr14, Arg127, Arg170. (G) Overexpressing BPGM significantly increased the protein level of p-CDK1-T 14 in tumor cells. Cells stably expressing BPGM (BPGM-OE) and its control cells (Ctrl) were used to detect protein level by western blotting. (H) 2,3-BPG treatment enhanced the phosphorylation of CDK1 at thr14 in tumor cells. The indicated concentration of 2,3-BPG was incubated with the lysate of trophoblasts and tumor cells for 30 minutes followed by western blotting. (I-J) RO-3306 treatment enhanced the phosphorylation of CDK1 at thr14 and reduced the phosphorylation of EZH2 at thr345 in tumor cells. The tumor cells were treated with the indicated concentration of RO-3306 for 12 hours followed by western blotting. (K) The model deciphers the role of BPGM in regulating BBOX1 and MMP9 expression. Error bar: mean ± SEM. P -values are labeled above the bar chart.

Article Snippet: The antibodies used included mouse antibody against β-actin (BM0627, Boster, Wuhan, China), rabbit antibody against BPGM (17173-1-AP, Proteintech), EZH2 (F0281, Selleck), phospho-EZH2 (Thr345) (TA3584S, Abmart, Shanghai, China), phospho-CDK1 (Thr14) (AP1465, Abclonal, Wuhan, China), ubiquitin (10201-2-AP, Proteintech), HIF1α (36169, Cell Signaling Technology, CST, Beverly, MA, USA), H3K4me3 (91264, Active Motif), H3K79me3 (cat 49-1020, Thermos Fisher), H3K9me3 (61014, Active Motif), H3K27me3 (91168, Active Motif) and Histone 3 (F0057, Selleck).

Techniques: Migration, Functional Assay, Expressing, Modification, Stable Transfection, Control, Western Blot, Amplification, Binding Assay, Phospho-proteomics, Concentration Assay, Incubation, Labeling

Journal: Neoplasia (New York, N.Y.)

Article Title: BPGM as an intrinsic brake to constrain metastasis through phospho-epigenetic-mediated carnitine biosynthesis suppression

doi: 10.1016/j.neo.2026.101299

Figure Lengend Snippet: Working model of BPGM-mediated metabolic-epigenetic regulation axis and its gatekeeper role in tumor metastasis. In low-metastatic tumors, higher oxygen levels activate KDM4A, which removes repressive H3K9me3 marks at the BPGM promoter, thereby promoting BPGM transcription. Elevated BPGM expression increases the production of 2,3-BPG, which stabilizes EZH2 and enhances SAM-dependent H3K27me3 deposition. This epigenetic remodeling leads to transcriptional silencing of BBOX1 , a key gene involved in carnitine biosynthesis, consequently suppressing fatty acid oxidation and inhibiting tumor metastasis. In contrast, under hypoxic conditions commonly found in high-metastatic tumors, KDM4A activity is diminished, resulting in the accumulation of H3K9me3 at the BPGM promoter and subsequent downregulation of BPGM expression. This disruption of the BPGM-mediated regulatory axis abrogates its anti-metastatic function. Notably, preclinical studies revealed that pharmacological inhibition of BBOX1 with Meldonium restores the metabolic-epigenetic barrier, effectively impeding metastatic progression.

Article Snippet: The antibodies used included mouse antibody against β-actin (BM0627, Boster, Wuhan, China), rabbit antibody against BPGM (17173-1-AP, Proteintech), EZH2 (F0281, Selleck), phospho-EZH2 (Thr345) (TA3584S, Abmart, Shanghai, China), phospho-CDK1 (Thr14) (AP1465, Abclonal, Wuhan, China), ubiquitin (10201-2-AP, Proteintech), HIF1α (36169, Cell Signaling Technology, CST, Beverly, MA, USA), H3K4me3 (91264, Active Motif), H3K79me3 (cat 49-1020, Thermos Fisher), H3K9me3 (61014, Active Motif), H3K27me3 (91168, Active Motif) and Histone 3 (F0057, Selleck).

Techniques: Expressing, Activity Assay, Disruption, Inhibition