Retro-2 treatment results in failure of the heterotypic fusion between autophagosomes and lysosomes.
HeLa cells stably expressing the autophagy marker microtubule-associated protein 1 light chain 3 (LC3) coupled to green fluorescent protein (GFP-LC3) were loaded with LysoTracker Red, allowing the identification and quantification of lysosomes only positive for LysoTracker Red, autophagosomes only positive for GFP-LC3, and autolysosomes positive for the merged Lysotracker Red/GFP-LC3 fluorescence signals. (A) z-stack confocal micrographs showing the rare presence of autophagosomes and the absence of autolysosomes in a control cell (left image), the classical strong presence of autolysosomes in a nutrient-starved cell (middle image), and the absence of autolysosomes, despite the presence of small and large autophagosomes, in a nutrient-starved cell subjected to the continuous presence of Retro-2 (1 μM) (right image). (B) Quantification of autophagosomes and autolysosomes per cell. *p < 0.01 compared to Control, **p < 0.01 compared to Autophagy-induced. (C) A representative western blot showing LC3 protein processing in control cells and nutrient-starved cells treated in the continuous presence of Retro-2 (1 μM), in the presence, or not, of chloroquine (CQ) (Left). Graph showing the quantification of LC3-II protein abundance (Right). *p < 0.01 compared to Control. The micrographs are representative of two independent experiments in duplicate. The white boxed areas show the region of high magnification in the adjacent images. Data were obtained by examining at least 30 cells for each condition in two independent experiments in duplicate. The western blot is representative of two separate experiments. Quantification in confocal images and western-blot quantification were performed using ImageJ software. Data are presented as the average ± SEM and were analyzed using the unpaired Student t test.