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. 2022 Mar 8;12(3):730–751. doi: 10.1158/2159-8290.CD-21-0385

Figure 4.

Figure 4. EP300 degradation rapidly disrupts MYCN expression and causes apoptosis. A, Kelly NB cells were treated with 1 μmol/L JQAD1, A485, or DMSO control for 12, 24, or 36 hours, prior to lysis and Western blotting for the markers of apoptosis: cleaved caspase-3 and cleaved PARP1. Actin is demonstrated as a loading control. Data is representative of three independent treatments and analyses in Kelly and NGP cells. B, Kelly cells were treated with 500 nmol/L JQAD1, A485, or DMSO control for 24 hours prior to External RNA Controls Consortium (ERCC)–controlled spike in RNA-seq. GSEA of RNA-seq results was performed with the MSigDB hallmarks dataset. n = 3 biological replicates and independent RNA extractions per treatment. C, Normalized RNA-seq gene expression of pro- and antiapopotic mRNA transcripts from Kelly cells treated as in B. Log10 transcript expression is shown, normalized against DMSO and ERCC controls. n = 3 biological replicates and independent RNA extractions per treatment. Bars, SEM. D, Nuclear lysates from Kelly cells were immunoprecipitated with anti-EP300, anti-CBP, or IgG control antibodies. WCL, whole-cell lysate. Data is representative of >3 independent co-IP/Western blots. E and F, Kelly cells were treated with DMSO control, A485 (0.5, 1 μmol/L), or JQAD1 (0.5, 1 μmol/L), followed by extraction of chromatin (E) or whole-cell lysates (F) and Western blotting. Total H3 is shown as a loading control. Data are representative of three independent biological replicates. See also Supplementary Fig. S4. NES, normalized enrichment score.

EP300 degradation rapidly disrupts MYCN expression and causes apoptosis. A, Kelly NB cells were treated with 1 μmol/L JQAD1, A485, or DMSO control for 12, 24, or 36 hours, prior to lysis and Western blotting for the markers of apoptosis: cleaved caspase-3 and cleaved PARP1. Actin is demonstrated as a loading control. Data is representative of three independent treatments and analyses in Kelly and NGP cells. B, Kelly cells were treated with 500 nmol/L JQAD1, A485, or DMSO control for 24 hours prior to External RNA Controls Consortium (ERCC)–controlled spike in RNA-seq. GSEA of RNA-seq results was performed with the MSigDB hallmarks dataset. n = 3 biological replicates and independent RNA extractions per treatment. C, Normalized RNA-seq gene expression of pro- and antiapopotic mRNA transcripts from Kelly cells treated as in B. Log10 transcript expression is shown, normalized against DMSO and ERCC controls. n = 3 biological replicates and independent RNA extractions per treatment. Bars, SEM. D, Nuclear lysates from Kelly cells were immunoprecipitated with anti-EP300, anti-CBP, or IgG control antibodies. WCL, whole-cell lysate. Data is representative of >3 independent co-IP/Western blots. E and F, Kelly cells were treated with DMSO control, A485 (0.5, 1 μmol/L), or JQAD1 (0.5, 1 μmol/L), followed by extraction of chromatin (E) or whole-cell lysates (F) and Western blotting. Total H3 is shown as a loading control. Data are representative of three independent biological replicates. See also Supplementary Fig. S4. NES, normalized enrichment score.