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anti ezh2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti ezh2
    Hypoxia increases expression of <t>EZH2,</t> 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 <t>anti-EZH2,</t> 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).
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    1) Product Images from "Interplay between nuclear survivin and the PRC2 complex and its impact on H3K27me3-directed transcriptional repression"

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

    Journal: Journal of Cell Science

    doi: 10.1242/jcs.264572

    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).
    Figure Legend 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).

    Techniques Used: Expressing, Western Blot, Cell Culture, Control

    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%.
    Figure Legend 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%.

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

    Related Articles

    Incubation:

    Article Title: Repurposing acetyldigitoxin as a potential EZH2 inhibitor for non-small cell lung cancer: a computational and experimental approach.
    Article Snippet: Equal amounts of protein were then separated by 10% sodium SDS-PAGE and transferred to PVDF membranes (Millipore). .. Subsequently, membranes were incubated overnight at 4 °C with the following primary antibodies (Cell Signaling Technology) diluted in blocking buffer: anti-EZH2 (1:1000, #5246), anti-Bax (1:1000, #5023), anti-Bcl-2 (1:1000, #15,071), anti-Caspase-3 (1:1000, #9662), anti-Cyclin D1 (1:1000, #2978), anti-H3K27me3 (1:1000, #9733) and anti-Histone H3 (total H3; 1:1000, #4499) for normalization, and anti-β-actin (1:2000, #4967) as a loading control. ..

    Article Title: Nuclear and cytoplasmic USP30-AS1 coordinately regulate breast cancer progression through HnRNPF/p21 and EZH2/c-Myc/p21 axes
    Article Snippet: .. Then the lysates were incubated with anti-EZH2 (CST, 5246S), anti-H3k27me3 (CST, 9733S), or anti-SPI1 (CST, 2266) antibodies, and magnetic beads (Invitrogen, #10004D) at 4 °C for 2 h. The DNA-protein complex was then washed three times using TE buffer, and DNA was purified and analyzed by real-time PCR. ..

    Article Title: Nuclear and cytoplasmic USP30-AS1 coordinately regulate breast cancer progression through HnRNPF/p21 and EZH2/c-Myc/p21 axes
    Article Snippet: The lysates were separated by SDS-PAGE and transferred to nitrocellulose membrane (Pall Corporation, #66485). .. The membranes were blocked and incubated with the following antibodies: anti-P21 (#2947S, CST), anti-HnRNPF (#67701-1-Ig, Proteintech), anti-GAPDH (#10442-1-AP, Proteintech), anti-α/β-Tubulin (#2148, CST), anti-EZH2 (#5246, CST), anti-H3k27me3 (#9733, CST), anti-c-Myc (#5605, CST), anti-CyclinD1 (#2978, CST). .. Protein bands were visualized using iBrightTM CL750 Imaging System (Thermo Fisher Scientific, USA).

    Article Title: Nuclear and cytoplasmic USP30-AS1 coordinately regulate breast cancer progression through HnRNPF/p21 and EZH2/c-Myc/p21 axes
    Article Snippet: RNA Immunoprecipitation was performed using MagnaRIP kit (Millipore, #17–700). .. Briefly, cells were lysed in RIP lysis buffer and the lysates were incubated with magnetic beads, anti-EZH2 (CST, #5246S), anti-HnRNPF (Sigma, #04–1462), or IgG antibodies. ..

    Blocking Assay:

    Article Title: Repurposing acetyldigitoxin as a potential EZH2 inhibitor for non-small cell lung cancer: a computational and experimental approach.
    Article Snippet: Equal amounts of protein were then separated by 10% sodium SDS-PAGE and transferred to PVDF membranes (Millipore). .. Subsequently, membranes were incubated overnight at 4 °C with the following primary antibodies (Cell Signaling Technology) diluted in blocking buffer: anti-EZH2 (1:1000, #5246), anti-Bax (1:1000, #5023), anti-Bcl-2 (1:1000, #15,071), anti-Caspase-3 (1:1000, #9662), anti-Cyclin D1 (1:1000, #2978), anti-H3K27me3 (1:1000, #9733) and anti-Histone H3 (total H3; 1:1000, #4499) for normalization, and anti-β-actin (1:2000, #4967) as a loading control. ..

    Control:

    Article Title: Repurposing acetyldigitoxin as a potential EZH2 inhibitor for non-small cell lung cancer: a computational and experimental approach.
    Article Snippet: Equal amounts of protein were then separated by 10% sodium SDS-PAGE and transferred to PVDF membranes (Millipore). .. Subsequently, membranes were incubated overnight at 4 °C with the following primary antibodies (Cell Signaling Technology) diluted in blocking buffer: anti-EZH2 (1:1000, #5246), anti-Bax (1:1000, #5023), anti-Bcl-2 (1:1000, #15,071), anti-Caspase-3 (1:1000, #9662), anti-Cyclin D1 (1:1000, #2978), anti-H3K27me3 (1:1000, #9733) and anti-Histone H3 (total H3; 1:1000, #4499) for normalization, and anti-β-actin (1:2000, #4967) as a loading control. ..

    Magnetic Beads:

    Article Title: Nuclear and cytoplasmic USP30-AS1 coordinately regulate breast cancer progression through HnRNPF/p21 and EZH2/c-Myc/p21 axes
    Article Snippet: .. Then the lysates were incubated with anti-EZH2 (CST, 5246S), anti-H3k27me3 (CST, 9733S), or anti-SPI1 (CST, 2266) antibodies, and magnetic beads (Invitrogen, #10004D) at 4 °C for 2 h. The DNA-protein complex was then washed three times using TE buffer, and DNA was purified and analyzed by real-time PCR. ..

    Article Title: Nuclear and cytoplasmic USP30-AS1 coordinately regulate breast cancer progression through HnRNPF/p21 and EZH2/c-Myc/p21 axes
    Article Snippet: RNA Immunoprecipitation was performed using MagnaRIP kit (Millipore, #17–700). .. Briefly, cells were lysed in RIP lysis buffer and the lysates were incubated with magnetic beads, anti-EZH2 (CST, #5246S), anti-HnRNPF (Sigma, #04–1462), or IgG antibodies. ..

    Purification:

    Article Title: Nuclear and cytoplasmic USP30-AS1 coordinately regulate breast cancer progression through HnRNPF/p21 and EZH2/c-Myc/p21 axes
    Article Snippet: .. Then the lysates were incubated with anti-EZH2 (CST, 5246S), anti-H3k27me3 (CST, 9733S), or anti-SPI1 (CST, 2266) antibodies, and magnetic beads (Invitrogen, #10004D) at 4 °C for 2 h. The DNA-protein complex was then washed three times using TE buffer, and DNA was purified and analyzed by real-time PCR. ..

    Real-time Polymerase Chain Reaction:

    Article Title: Nuclear and cytoplasmic USP30-AS1 coordinately regulate breast cancer progression through HnRNPF/p21 and EZH2/c-Myc/p21 axes
    Article Snippet: .. Then the lysates were incubated with anti-EZH2 (CST, 5246S), anti-H3k27me3 (CST, 9733S), or anti-SPI1 (CST, 2266) antibodies, and magnetic beads (Invitrogen, #10004D) at 4 °C for 2 h. The DNA-protein complex was then washed three times using TE buffer, and DNA was purified and analyzed by real-time PCR. ..

    other:

    Article Title: Interplay between nuclear survivin and the PRC2 complex and its impact on H3K27me3-directed transcriptional repression
    Article Snippet: Samples were counterstained in 1 μg/ml DAPI or NucBlue (Thermo Fisher Scientific) in PBS (10 min, RT) to stain nuclei, washed again in PBS then mounted using Mowiol mounting medium (Sigma-Aldrich, 81381).

    Western Blot:

    Article Title: Accessory subunits of PRC2 mimic H3K27me3 to restrict the spread of Polycomb domains.
    Article Snippet: Densitometry of the bands was measured using BioRad Image Lab. .. The antibodies used for immunoblotting included: anti-GAPDH (Proteintech 10494-1-AP, 1:4000), anti-H3 (Abcam #Ab1791, 1:100000), anti-H3K27me1 (Active Motif 61015, 1:2000), anti-H3K27me2 (Abcam ab24684, 1:1000), anti-H3K27me3 (Active Motif 61017, 1:2500), anti-EZH2 (Cell Signaling 5246S, 1:1000), anti-SUZ12 (Cell Signaling 3737, 1:1000), anti-EED (Cell Signaling 85322, 1:1000), anti-LCOR (Merck #ABE1367, 1:250), anti-JARID2 (Cell Signaling 13594S, 1:1000), anti-LAMINB1 (Abcam ab16048, 1:10000), anti-mouse-HRP-conjugated (Jackson ImmunoResearch #715-035-150, 1:5000), and anti-rabbit HRP-conjugated (Santa Cruz Biotechnology #sc-2357, 1:5000). .. Approximately 3–4 × 104 cells were cytospun onto glass slides and allowed to air-dry slightly before fixation in 4% paraformaldehyde solution (PFA) in PBS (Santa Cruz sc-281692) for 15 min at room temperature.

    Lysis:

    Article Title: Nuclear and cytoplasmic USP30-AS1 coordinately regulate breast cancer progression through HnRNPF/p21 and EZH2/c-Myc/p21 axes
    Article Snippet: RNA Immunoprecipitation was performed using MagnaRIP kit (Millipore, #17–700). .. Briefly, cells were lysed in RIP lysis buffer and the lysates were incubated with magnetic beads, anti-EZH2 (CST, #5246S), anti-HnRNPF (Sigma, #04–1462), or IgG antibodies. ..



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    Image Search Results


    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).

    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

    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%.

    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

    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).

    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

    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).

    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

    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).

    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

    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%.

    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

    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.

    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

    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.

    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