Figure 4
P300 and CBP dual knockdown inhibits CDK1 and cyclin B1 expression and H3K18 and H3K27 acetylation, but not H3K9 acetylation. (A, B) Cell viability following CBP and p300 siRNA treatment as determined by WST-8 assay (A) and BrdU incorporation assay (B). Cells were transfected with CBP- and p300-specific siRNAs or negative control siRNA for 48 h and cell viability was assessed every 24 h through 72 h (WST-8 assay) (A) or at 72 h (BrdU incorporation assay) (B). Error bars represent mean ± SD. *p < 0.05 vs controls treated with negative control siRNA by one-way ANOVA with post hoc Dunnett's test (A) and t-test (B). (C) Effects on CBP, p300, and PCAF expression and expression of G2/M cell cycle regulatory molecules after p300 and CBP siRNA treatment. Effective knockdown of CBP and p300 by treatment with CBP- and p300-specific siRNAs was confirmed at the protein level. PCAF expression was increased by p300 and CBP gene silencing. Expression of G2/M cell cycle regulatory molecules were suppressed. Cancer cells were treated with CBP- and p300-specific siRNAs or negative control siRNA for 72 h. (D) Effects of silencing both p300 and CBP genes on histone H3 acetylation. Effects on acetylation of H3K9, H3K18, and H3K27 were assessed. Acetylated H3K18 (H3K18Ac) and H3K27 (H3K27Ac) were downregulated by CBP and p300 gene silencing, while acetylated H3K9 (H3K9Ac) was not affected. (E) Effects of C646 treatment on histone H3 acetylation and CBP, p300, and PCAF expression in PSN1 and MIAPaCa2 cells. Cancer cells were treated with C646 for 72 h at each concentration (20 and 40 µM). At a C646 concentration of 40 µM, which sufficiently inhibited histone acetylation, expression of these HAT molecules was downregulated. (F) Effects of CBP, p300, and PCAF mRNA levels following C646 treatment. mRNA expression of p300, CBP, and PCAF were inhibited after 48-h treatment with 30 µM C646 treatment in PSN1 and MIAPaCa2 cells.
