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anti survivin  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti survivin
    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 <t>and</t> <t>anti-survivin</t> 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).
    Anti Survivin, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/survivin/pmc13035274-112-8-9
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    Images

    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

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    Incubation:

    Article Title: PHI-501, a dual inhibitor of RAF and DDR1/2, overcomes MAPK drug resistance in Melanoma.
    Article Snippet: Proteins were then transferred to a polyvinylidene fluoride (PVDF) membrane (Merck Millipore, Darmstadt, Germany) and blocked with 5% BSA in TBST (0.1% Tween-20 in TBS) for 1 h at room temperature. .. The membranes were incubated overnight at 4 °C with primary antibodies diluted in 5% BSA in TBST as indicated in the text: p-DDR1 (Cell Signaling Technology, CST14531), p-DDR1/2 (R&D Systems, MAB25382), p-AKT (Cell Signaling Technology, CST4058), AKT (Cell Signaling Technology, CST9272), p-MEK (Cell Signaling Technology, CST9121), p-ERK (Cell Signaling Technology, CST9101), ERK (Cell Signaling Technology, CST9102), Cyclin D1(Cell Signaling Technology, CST2978), and Survivin (Cell Signaling Technology, CST2808). .. After washing, the membranes were incubated with goat anti-rabbit or goat antimouse secondary antibodies conjugated to horseradish peroxidase (Invitrogen, Carlsbad, CA, USA). diluted in 1× TBS for 1 h, and immunoreactivity was detected using an enhanced chemiluminescence substrate for horseradish peroxidase (HRP) (Elpisbio, Daejeon, Korea;



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    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 <t>and</t> <t>anti-survivin</t> 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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    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: 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

    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).

    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: 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: Staining, Two Tailed Test, Western Blot, Fractionation, Expressing, Control

    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

    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).

    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

    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 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

    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 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

    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: 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

    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).

    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

    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: 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

    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.

    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