Fig. 5. Stimulation of autophagy by transient expression of wild-type (WT) Sirt1. HCT116 cells were transfected with GFP-LC3 along with an empty vector, wild-type Sirt1, or with a deacetylase-inactive mutant (HY) of Sirt1. Autophagy was assessed by counting the number of GFP-LC3 punctae per cell. All measurements were done under fed conditions.

Fig. 6. Reduced autophagy in Sirt1-^/- mouse embryonic fibroblasts (MEFs). High-power view of WT or Sirt1-^/- MEFs transfected with GFP-LC3 under fed or starved conditions is shown. In WT cells, starvation induces an increase in GFP-LC3 punctae.

Fig. 7. Sirt1-^/- MEFs have reduced levels of autophagy after stimulation with rapamycin. GFP-LC3-transfected wild type (+/+) or Sirt1-^/- MEFs were analyzed with or without rapamycin treatment.

Fig. 8. Effect of Sirt1 knockdown on autophagy in HeLa cells. (a) HeLa cells were transfected with a nontargeting RNAi or with one directed against Sirt1 and then maintained in fed conditions or shifted to starvation medium. Western blot (WB) analysis demonstrates the efficacy of Sirt1 knockdown. (b) HeLa cells with knockdown Sirt1 have reduced GFP-LC3 punctae formation under starved conditions.

Fig. 9. Colocalization of Sirt1 and Atg7. HeLa cells were transfected with FLAG-Sirt1 and myc-tagged Atg7. Twenty-four hours after transfection, cells were fixed and assayed by indirect immunofluorescence. (A) Sirt1 expression as assessed by using an anti-Sirt1-specific antibody in green. Transfected cells stain bright green, whereas nearby nontransfected cells are fainter and demonstrate endogenous Sirt1 expression. (B) An anti-myc-specific antibody demonstrated in red. (C) Overlap fluorescence demonstrated in yellow.

Fig. 10. Nicotinamide increases acetylation of Atg8. HeLa cells were transfected with a His-tagged Atg8 construct, and where indicated, they were harvested 14 h after nicotinamide treatment (10 mM). Levels of acetylation were assessed by direct Western blotting (WB) using an antibody directed against acetyllysine residues.

Fig. 11. Sirt1 can deacetylate Atg8 in vitro. His-tagged Atg was purified from transfected HeLa cells and incubated in the presence or absence of 10 mM NAD with either WT Sirt1 protein or a deacetylase-inactive (HY) mutant of Sirt1. Levels of acetylation were determined by Western blotting (WB) using an antibody that recognizes acetyllysine. Total Atg8 protein levels used in the reaction are shown below.

Fig. 12. Sirt3 expression does not reduce Atg5 acetylation. HeLa cells were transfected with myc-tagged Atg5 or an empty vector along with either myc-tagged Sirt3 or its corresponding empty vector. Levels of acetylated Atg5 were determined by first immunoprecipitating (IP) with an antibody directed against acetyllysine residues and then Western blot (WB) analysis using an antibody against the myc epitope. The mobility of myc-Sirt3 is directly below that of myc-Atg5.

Fig. 13. Activation of AMPK in myocardium of Sirt1-^/- neonates. Levels of phosphorylated AMP kinase (p-AMPK) (Thr-172) were determined from heart lysate of a 15-h-old WT and Sirt1-^/- littermates. Data are from a single pair of littermates but are representative of three separate experiments.

Fig. 14. Acetylation of WT and mutant Atg5. A site-directed mutant of Atg5 at position 130 (lysine to arginine substitution) was constructed. This residue has been implicated in forming a covalent bond with Atg12. WT and Atg5_K130R were transfected into HeLa cells, and overall levels of Atg5 expression and acetylation were determined. For unclear reasons, Atg5_K130R appeared to have higher expression levels than the WT Atg5, which we interpret as most likely caused by changes in overall protein stability after this amino acid substitution. Nonetheless, adjusted for overall protein levels, the amount of acetylation appears to be similar for these two Atg5 constructs.