Figure 1.

Resveratrol promotes an increase of autophagosomes. U2OS, HeLa, G361 and MCF-7 cells were treated with 64 µM resveratrol (Res), nutrient-free medium (EBSS) or control medium (CM) for 3 h. Bafilomycin A₁ (Baf A₁) was added after 2 h as indicated (+). LC3-II protein abundance was determined and normalized over tubulin or gapdh. The quantification of one representative experiment (n = 3 for each cell line) is shown (A). U2OS cells were transiently transfected with GFP-LC3 (B) or GFP-WIPI-1 (C) and treated with 64 µM resveratrol (Res), nutrient-free medium (EBSS) or control medium (CM) for 3 h and images were acquired by confocal microscopy. The number of GFP-LC3 puncta per cell was determined from 33–36 individual cells (n = 3) by using Z-stack projections and ImagePro Plus 4.1 analysis software (B, upper panel). The number of GFP-WIPI-1 puncta-positive cells per treatment was determined from 400 individual cells (n = 4) by fluorescent microscopy (C, upper panel). The results were expressed as mean ± SD p-values: *p < 0.05; **p < 0.01; ***p < 0.001; ns: not significant. Scale bars: 20 µm. Stable GFP-WIPI-1 U2OS cells were treated with control medium (CM), 64 µM resveratrol (Res) or nutrient-free medium (EBSS) in the presence (+) or absence (-) of bafilomycin A₁ (Baf A₁). For EM analysis, ultrathin resin sections of different samples were used and representative images are shown (D). Scale bars: 400 nm. The number of multilayered autophagosomal vesicular structures (AVi) per square micrometer was determined for each treatment (E).