Figure 1.

Sirtuin-1 (Sirt-1) induces autophagic vacuolization and has no effects on autophagosome–lysosome fusion. (a–c) Detection of autophagic vacuoles induced by Sirt-1 overexpression and modulation by EX527 in wild type (WT) HCT 116. Cells were co-transfected with a plasmid for the expression of GFP-LC3 together with an empty control vector (pcDNA3) or a Sirt-1-encoding plasmid for 24 h, and then cultured for 6 h in the absence or presence of 100 μM EX527. Representative microphotographs are reported in panel a, and the percentage of cells exhibiting the accumulation of GFP-LC3 in puncta (GFP-LC3^vac) is reported in panel b (mean±S.E.M., n=3, ^*P<0.05). The abundance of Sirt-1, p53 acetylated on Lys382 (p53 Ac Lys382), total p53 as well as LC3 maturation was determined by immunoblotting. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels were assessed to ensure equal loading of lanes. (c) Representative of three independent experiments. Numbers next to bands indicate MW (kDa). (d–f) The effect of bafilomycin A1 (BafA1) on Sirt-1-induced autophagic vacuolization. GFP-LC3-expressing HeLa cells were transfected with the pcDNA3 control vector or with a Sirt-1-encoding plasmid for 24 h, treated with 1 nM BafA1 for further 6 h and finally processed to assess the colocalization between GFP-LC3 and LAMP-2. (d) Representative confocal microphotographs and colocalization profiles within the area of interest (defined by the α → ω arrow). (e) Columns depict the percentage of colocalization between GFP-LC3 and LAMP-2, as quantified in at least 50 cells for each experimental condition (mean±S.E.M., n=3, ^*P<0.05). (f) The percentage of GFP-LC3^vac cells (mean±S.E.M., n=3, ^*P<0.05; n.s, nonsignificant). (g, h). Autophagic effects of SIR2.1 overexpression in C. elegans. (g) Representative images of WT or SIR2.1-overexpressing (geIn3 genotype; see Materials and Methods) C. elegans cells exhibiting DsRed::LGG1 puncta. (h) Columns depict mean pixel intensity of DsRed::LGG1 (mean±S.E.M., n=3, ^*P< 0.05)