Human colon carcinoma HCT 116 cells were transfected with an siRNA specific for SIRT1 and then retransfected with a GFP-LC3-encoding plasmid, cultured in complete medium for 24 h and left untreated for 4 h as a control for resveratrol or spermidine treatment. For fluorescence microscopy determinations, cells cultured on coverslips were fixed in paraformaldehyde (4% wt/vol) for 15 min at RT, washed three times in PBS, and mounted with mounting medium (Vectashield). Confocal fluorescent images were captured using a confocal fluorescence microscope (TCS SP2; Leica) fitted with an Apochromat 63× 1.3 NA immersion objective. Images were acquired with a camera (DFC 350 FX 1.8.0; Leica) using LAS AF software (Leica) and processed with Photoshop (CS2; Adobe) software. Specifically, picture processing involved cropping of representative areas and linear adjustments of contrast and brightness and was performed using Photoshop (with equal adjustment parameters for all pictures); no explicit γ correction was used. Image: Figure 1A, bottom left panel, in Morselli et al. J Cell Biol 192: 615-629