FIGURE 4.

SIRT1 activity and the mitochondrial content in NAM-treated cells. A and B, cells incubated with 5 mm NAM for indicated time periods were applied to Western blotting for acetylated or total H3 or SIRT1 protein (A) and acetylated or total p53 or Erk protein. The protein bands were quantified by densitometry, and the values relative to those in the control cells (0 h) were averaged (from three different blots) and plotted. C, in vivo SIRT1 activity measured using Fluor-de-Lys assay kit. Each bar represents the mean ± S.E. of six independent experiments. *, p is < 0.02 compared with the 0 h control (−). D, cells cultured in the presence of 5 or 20 mm NAM were collected at the indicated time points, stained with nonyl acridine orange, and applied to flow cytometry to determine mitochondrial content. The quantities relative to those of the 0 h control from three biological repeats were averaged and plotted. A and D, *, ^#, p < 0.05; ^##, p < 0.01 compared with the day 0 control by ANOVA test (Dunnett's test).