Quantification of LC3 puncta and autophagic flux

To quantify the number of autophagosomes, the LC3 fluorescent images were first processed by ImageJ (National Institutes of Health) and then analyzed by using MetaMorph (Molecular Devices, Transflour). Usually, cells show low basal level of autophagy under normal culture condition. We thus used computerized software image analysis program (Top Hat algorithm of MetaMorph) to analyze LC3 puncta diameter. In Figure 5, cells displaying more than 30 intense LC3 puncta of 1 to 3 μm were counted as autophagy-positive cells. At least 150 cells were analyzed for each condition. ImageJ software was also used to quantify the intensity ratio of GFP-LC3 to RFP-LC3ΔG. We marked the edges of individual cells and quantified the intensity of GFP and RFP signals in each marked area. At least 150 cells were analyzed for each condition. The images of GFP:RFP ratio were processed by the ImageJ Ratio Plus plugin to display as a pseudo-color imaging.