(A) Western blot analysis of p53 and BAX from mictochondrial fractions, 12 h after 2-ME withdrawal, in wild-type and SIRT1−/− mES cells. Mitochondrial fractions were prepared for p53 and BAX western blotting as described in “Experimental Procedures”. The results of HSP-70 analysis are shown as a marker for mitochondrial fractions. The results of β-actin and PCNA analysis are shown to compare the extend to which the cysolic and nuclear fractions are contaminated. (B) Apoptosis of wild type, SIRT1−/− mES cells and SIRT1−/− mES cells infected with control or SIRT1 expressing lentiviral particles 60 h after 2-ME withdrawal as detected by FACS after Annexin V staining. Error bars represent SD of triplicate experiments. (C) Expression levels of p53 and BAX in scrambled shRNA, p53 shRNA or BAX shRNA expressing wild type mES cells, as detected by western blot analysis. (D) Apoptosis of scrambled shRNA, p53 shRNA or BAX shRNA expressing wild type mES cells 60 h after 2-ME withdrawal as detected by FACS after Annexin V staining. Error bars represent SD of triplicate experiments. Results shown in A–D are for 1 of 3 reproducible experiments with R1 mES cells.