Confocal imaging of mCherry-GFP-LC3 vesicles, mCherry-WDFY vesicles, and immunofluorescence staining for LAMP1 or LAMP2 vesicles shows a quantification of autophagosomes, autolysosome, and phagophores. (A) mCherry-GFP-LC3 protein is useful for distinguishing between autophagosomes and autolysosomes because of the difference of pH in the lumens. mCherry and GFP proteins are stable in the neutral pH of the lumen of autophagosomes, whereas GFP protein is acid-labile and mCherry protein is acid-stable in the autolysosome. (B) Nutrient-deprived HepG2 cells show more yellow-stained autophagosomes and red-stained autolysosomes in confocal microscopy. (C) The total number of autophagic vesicles increases in cells without nutrients (starvation). (D) The ratio of the number of autolysosome spots (mCherry+GFP-) to total spots (autophagosomes, mCherry+GFP+ and autolysosomes, mCherry+GFP-) is proportional to the fusion of autophagosomes with lysosomes. Treatment with Rapamycin, an inducer of autophagy, shows values similar to starvation (EBSS, Earle’s balanced salt solution) and treatment with compound X inhibits the fusion of autophagosomes with lysosomes. Quantitative data are presented as means ± SD from three independent experiments. (E) HepG2 cells expressing mCherry-WDFY (PI3P-binding protein) and GFP-LC3 indicate phagophores (red, mCherry+GFP-), early autophagosomes (yellow, mCherry+GFP+), and late autophagosomes (green, mCherry-GFP+). Treatment with SAR405, an inhibitor of Vps34, decreases the number of phagophores. Quantitative data are presented as means ± SD from three independent experiments. (F) Immunofluorescence staining of HepG2 cells expressing mCherry-GFP-LC3 with antibodies to LAMP1 or LAMP2 lysosomal membrane protein indicates autophagosomes (yellow, mCherry+GFP+) and autolysosomes (red spots in a far-red vesicle, mCherry+GFP- spots in a vesicle containing LAMP1/2). (G) HepG2 cells expressing mCherry-GFP-LC3 were observed using confocal microscopy and the size of autolysosome vesicles was calculated by measuring the diameter. Treatment with Baf A1 inhibits degradation of cytosolic compartments derived from autophagosomes in lysosomes and increases in the number of enlarged autolysosomes. Quantitative data are presented as means ± SD from three independent experiments.