Figure 2
BRD8 knockdown induces p53-dependent apoptosis in p53+/+ cells and G2 arrest in p53−/− cells. Apoptosis in HCT116 p53+/+ (a) and HCT116 p53−/− (b) cells were quantified by FACS analysis of Annexin V and PI double staining 72 h post transfection with two independent siRNAs targeting BRD8 (siBRD8-35 and siBRD8-36) (or control siRNA (Ctrl)). Immunoblot showing the cleaved PARP in HCT116 p53+/+ (c) and HCT116 p53−/− (d) cells in BRD8 depleted cells. Numbers represent densitometric analysis normalized to actin. (e) Crystal violet staining assay of HCT116 p53−/− cells at 24, 48, 72 and 96 h post transfection with siRNAs targeting BRD8 (siBRD8-35 and siBRD8-36) or control siRNA (CT), the cells were fixed and stained with crystal violet. Crystal violet stain was dissolved and optical absorbance was measured at 590 nm (OD590). Three independent experiments were performed; the mean ± standard deviation is shown. (f) Cell cycle distributions of HCT116 p53−/− following BRD8 knockdown for 72 h and analyzed using flow cytometry. Data are the mean ± SD from three independent experiments. An ANOVA test followed up with a Dunnett analysis was used to compare each mean to its relative control. *P ≤ 0.05; **P ≤ 0.01; ns = non-significant compared to relative control.
