Figure 9. Sirt3 binds, deacetylates, and activates Foxo3a.

(A) Lysate of cells overexpressed with Flag or Flag.Sirt3 was subjected to immunoprecipitation with Flag-M2 agarose beads. The resulting complex was analyzed by Western blotting with anti-Foxo3a antibody. (B) Lysate of cells infected with Ad.Sirt3 virus was subjected to immunoprecipitation with anti-Foxo3a antibody, and the resulting complex was analyzed by Western blotting with anti-Sirt3 antibody. (C) Sirt3 deacetylates Foxo3a. Cells were overexpressed with Flag.Foxo3a and then treated with H₂O₂ to induce protein acetylation. Flag.Foxo3a was immunoprecipitated and subjected to deacetylation with Sirt3 or Sirt1 in the presence or absence of NAD in the buffer. Protein acetylation was determined by Western blotting with anti–Ac-K and anti-Flag antibodies. (D) Cytoplasmic and nuclear fractions of cardiomyocytes infected with Ad.Sirt3 or Ad.Smut viruses were prepared and analyzed by Western blotting with Foxo3a antibody. Histone 3 and GAPDH were utilized as nuclear and cytoplasmic markers, respectively. (E) Confocal analysis of Foxo3a localization (red) in cardiomyocytes infected with Ad.Sirt3 or Ad.Smut and treated with PE. Positions of nuclei were determined by DAPI stain (blue). Original magnification, ×1,000. (F) Overexpression of Sirt3 activates Foxo3a-dependent promoters. Neonatal rat cardiomyocytes were infected with viruses synthesizing Sirt1, Sirt3, or a mutant protein. The next morning cells were transfected with a Foxo3a expression plasmid and a Foxo3a responsive/luciferase reporter plasmid in different combinations as indicated. Luciferase activity was determined 48 hours after transfection. Sirt3 overexpression significantly activated Foxo3a-dependent promoter. Mean ± SEM, n = 4.