Figure 2

Resveratrol (Resv) triggers autophagic vacuolization and fails to inhibit the fusion of autophagosomes with lysosomes. (a) Detection of autophagic vacuoles induced by Resv and modulation by EX527 in wild-type (WT) HCT 116. Cells were transfected with a plasmid encoding GFP-LC3 for 24 h and cultured for additional 6 h in the absence or presence of 100 μM Resv and/or 100 μM EX527. The percentage of cells showing the accumulation of GFP-LC3 in cytoplasmic puncta (GFP-LC3^vac) is reported (mean±S.E.M., n=3, ^*P<0.05). (b) The levels of Sirtuin-1 (Sirt-1), p53 acetylated on Lys382 (p53 Ac Lys382), total p53 as well as LC3 maturation were determined by immunoblotting. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) abundance was monitored to ensure equal loading of lanes. These data are representative of three independent experiments. Numbers next to bands illustrate MW (kDa). (c–e) Effect of bafilomycin A1 (BafA1) on Resv-induced autophagy. GFP-LC3-expressing HeLa cells were treated with Resv and/or BafA1 for 6 h (unless otherwise indicated) and then processed to assess the colocalization between GFP-LC3 and LAMP-2. Representative confocal microphotographs are shown in panel c, together with the colocalization profiles within the area of interest (defined by the α → ω arrow). (d) Columns illustrate the percentage of colocalization between GFP-LC3 and LAMP-2 (mean±S.E.M., ^*P<0.05), as quantified in at least 50 cells for each experimental condition. (e) The kinetics of GFP-LC3 redistribution was quantified by conventional fluorescence microscopy (mean±S.E.M., n=3, ^*P<0.05). (f) LC3 maturation in WT HCT 116 treated with Resv and/or leupeptin (Leu) for the indicated time. GAPDH levels were assessed to ensure equal loading of lanes. Data are representative of three independent experiments. Numbers next to bands indicate MW (kDa). (g, h) The effects of Sirt-1 knockdown on Resv-induced autophagy. WT HCT 116 cells transfected with the indicated siRNAs were re-transfected with a plasmid for the expression of GFP-LC3 for 24 h and then cultured in the presence or absence of 100 μM resveratrol for further 6 h. (g) Columns depict the percentage of GFP-LC3^vac cells (mean±S.E.M., n=3, ^*P<0.05). The abundance of Sirt-1, p53 acetylated on Lys382 (Ac Lys382 p53), total p53, and LC3I/II was determined by immunoblotting. GAPDH levels are shown as control for the equal loading of lanes. (h) Representative of three independent experiments. Numbers next to bands depict MW (kDa). (i) Induction of autophagy by Resv and its dependence on SIRT-2.1 in C. elegans. The effects of 100 μg/ml Resv were assessed either on WT animals or on sir-2.1(ok434) deletion mutants expressing the DsRed::LGG-1 transgene. Columns depict mean pixel intensity of DsRed::LGG1 (mean±S.E.M., n=3, ^*P<0.05)