Basal autophagy occurs in resting platelets. (A) Western blot analysis of LC3II in unstimulated human platelets treated with either NH₄Cl (20 mM) or chloroquine (50 µM) to inhibit lysosomal activity. Two exposure times, short and long, are shown. Note that LC3I is visible only with longer exposures in this experiment. (B) GFP-LC3 puncta in unstimulated platelets without and with NH₄Cl treatment (20 mM, 2 h). Samples were visualized for DIC and GFP fluorescence (GFP-LC3). Ten images (17∼74 platelets/field) were obtained at random and quantified for each condition. Platelets containing GFP-LC3-positive puncta are 21 ± 8% and 64 ± 10% without and with NH₄Cl treatment, respectively. Statistical significance was evaluated with the Student t test. (C) Western blot analysis of LC3II in human platelets pretreated for 1 h with cycloheximide (100 µg/mL) or puromycin (10 µg/mL), in combination with pretreatment of NH₄Cl (20 mM, 1 h) or protease inhibitor cocktail (1 h). The images shown are representative of at least 3 independent experiments.