Fig. 2: PPI didn’t inhibit the fusion of autophagosomes and lysosomes in neutral pH condition.

a AGS cells were either untreated or treated with PPI (60–140 μg/ml) for 24 h in pH 7.4 condition in the absence or presence of classical autophagic flux inhibitor baf-A1 (100 nM). The indicated protein levels were analyzed by western blot. b AGS cells were transiently infected with GFP-mRFP-LC3B adenoviral particles for 48 h and subsequently treated with PPI (120 μg/ml), baf A1 (100 nM), Torin 1 (500 nM) for 48 h in pH 7.4 condition. The change of both green and red fluorescence was observed using a confocal microscope. Scale bar: 20 μm. Lower panel, the numbers of acidified autophagosomes (GFP⁻RFP⁺) versus neutral autophagosomes (GFP⁺RFP⁺) per cell in each condition were quantified. Data were presented as mean ± SD from three independent experiments (n.s. not significant; **p < 0.01, identified by two-way ANOVA with Dunnett’s multiple comparison test). c AGS cells were either untreated or treated with PPI (60–140 μg/ml) for 24 h in pH 7.4 condition in the absence or presence of rapamycin (1 μM) or baf-A1 (100 nM), then stained with LysoTracker Red DND-99 dye (50 nM for 15 min) for FACS analysis. Representative results of three independent experiments were shown. d, e qRT-PCR analysis of various V-ATPase subunits (d) and lysosomal genes (e) in AGS cells treated with 100 and 120 μg/ml PPI for 48 h in pH 7.4 condition. Data were presented as mean ± SD. f Different cancer cells were either untreated or treated with 100 μg/ml PPI for 48 h in pH 7.4 condition, and then stained with LTR for FACS analysis. Representative results of three independent experiments were shown. g AGS and MKN45 cells were pretreated with indicated concentrations of PPI for 24 h in pH 7.4 condition, and then incubated with or without rapamycin (1 μM) for another 24 h. The change pattern of SQSTM1 protein was determined by western blot