FIG. 12.

Redundant function of SIRT3 and SIRT1 of protecting cells from apoptosis. (A) SIRT3- and SIRT1-mediated deacetylation of Ku70 enhances its binding to Bax. Flag-Ku70 was synthesized in vitro, captured on Flag beads, and then acetylated by PCAF. Acetylated Flag.Ku70 was subjected to deacetylation by SIRT1, SIRT3, or the mt protein. The acetylated and deacetylated Flag.Ku70 was then incubated with HeLa cell extract in CHAPS buffer overnight. Flag.Ku70 beads were separated from the extract and tested for Bax coprecipitation. Beads were also tested for the acetylation status of Ku70 by Western blotting using appropriate antibodies. Note that Bax was pulled down by Ku70 that was deacetylated by either SIRT1 or SIRT3 but not by the acetylated Ku70. (B) SIRT1 was knocked out (KO) from wt SIRT3- or mt SIRT3-expressing stable cell lines, using SIRT1-specific siRNA. The blot shows Western analysis of SIRT1 expression levels in control and KO cells. (C) SIRT1 KO sensitized mt SIRT3-expressing cells, but not the wt SIRT3-expressing cells, to MNNG-mediated cell death. Values are means ± standard errors of four experiments. (D) Representative density blots of FACS analysis of cells treated with MNNG (500 μM). Cell death was monitored 3 h later.