FIG. 10.

SIRT3 blocks Ku70 acetylation during stress. (A) Ku70 was acetylated during genotoxic and oxidative stress. Cells were transiently expressed with Flag-Ku70 and treated with MNNG (500 μM) or H₂O₂ (500 μM). Cells were harvested at indicated time intervals after treatment, and Flag.Ku70 was immunoprecipitated. Flag.Ku70 beads were analyzed by Western blotting using antiacetyllysine and anti-Ku70 antibodies. (B) Quantitative representation of Ku70 acetylation by MNNG and H₂O₂ treatment of cells. Values are means of three separate experiments. (C) HeLa cells stably expressing either wt or mt SIRT3 were treated with MNNG for 2 h. The endogenous Ku70 was immunoprecipitated and analyzed by Western blotting as in panel A. (D) Knockdown of SIRT3 increases Ku70 acetylation during genotoxic stress. SIRT3 was knocked down from SIRT3-expressing stable cells by use of SIRT3-specific siRNA. Scrambled RNA was used as a negative control. Cells were treated with MNNG for 2 h, and the level of Ku70 acetylation was determined by Western blotting.