Figure 7.

SIRT6 or CHD4 depletion impairs repair protein loading and compromises cell survival. (A) HeLa cells were transfected with NS (non-specific), SIRT6 or CHD4-specific siRNA for 48 h in the presence or absence of DOX. Chromatin fractions were extracted and analyzed by western blotting. (B and C) HeLa cells were transfected with NS, CHD4 or SIRT6-specific siRNA for 48 h. Cells were exposed to 10 Gy irradiation (IR), and then fixed at 1, 3 or 6 h post-IR and observed under a confocal microscope. The numbers of PRA (B) and BRCA1 (C) foci per cell were quantified. The data represent the means ± SEM (n = 100, *P < 0.05, **P < 0.01, ***P < 0.001). (D and E) HeLa cells stably expressing NS, SIRT6 or CHD4 specific siRNA were treated with 20 or 40 μM VP16 for 2 h and then washed free of the drug. The cells were then cultured in a six-well plate for 2 weeks, and clone formation was analyzed by crystal violet staining. (F) HeLa cells stably expressing NS, SIRT6 or CHD4-specific siRNA were exposed to 3 Gy IR. The cells were then cultured in a 6-well plate for 2 weeks, and clone formation was analyzed by crystal violet staining. (G) A Flag-SIRT6-FL, Flag-SIRT6-ΔC mutant, Flag-SIRT6-Core or Flag-SIRT6-ΔN mutant plasmid was transfected into shSIRT6 HeLa cells. The cells were then treated with 40 μM VP16 for 2 h and washed free of the drug. Then the cells were cultured in a 6-cm plate for 2 weeks and clone formation was analyzed by crystal violet staining. The data represent the means ± SEM (n = 3, *P < 0.05, **P < 0.01, ***P < 0.001).