Figure 6
BRD8 knockdown causes spontaneous DNA damage and decreases chromatin- bound H4K16 acetylation. (a) Detection of DNA damage foci in BRD8 knockdown cells. HCT116 p53+/+ cells were transfected with BRD8-targeting (siBRD8-35) or control (Ctrl) siRNAs. Cells were fixed and γH2A.X foci (red) and BRD8 (green) localization were monitored by immunostaining. Nuclei were counterstained with DAPI. Scale bar is 20 μm. (b) The histogram represents the percentage of cells with more than 10 γH2A.X foci. Total cell extracts were subjected to immunoblot assays using the indicated antibodies 48 h following knockdown with BRD8-targeting (siBRD8-35 and siBRD8-36) or control (Ctrl) siRNAs in HCT116 p53+/+ cells (c) and with BRD8-targeting (shBRD8) or control (Ctrl) shRNAs in HCT116 p53−/− cells (d). (e) Total histone extracts of HCT116 p53+/+ were subjected to immunoblot assays using indicated antibodies 48 h post transfection with BRD8-targeting (siBRD8-35 and siBRD8-36) or control (Ctrl) siRNAs. (f) Densitometric analysis of Western blot with average intensity values from three independent experiments. Intensities were calculated using Image Lab and normalized to the intensities of total histone H4. The mean ± SD from three independent experiments are shown. An ANOVA test followed up with a Dunnett analysis was used to compare each mean to its relative control. *P ≤ 0.05; compared to control.
