Fig. 3. PPI upregulated SQSTM1 mRNA via NFE2L2.

a AGS, HGC27, and HeLa cells were treated with 60 and 100 μg/ml PPI for 48 h in both pH 6.5 and pH 7.4 conditions, respectively. The SQSTM1 mRNA levels were assessed (**p < 0.01, for each cell line using one-way ANOVA with Dunnett’s multiple comparisons test). b AGS cells were treated with PPI for 48 h in both pH 6.5 and pH 7.4 conditions, and then stained with DCFHDA (5 μM) for 30 min. Intracellular ROS was reflected by the fluorescence intensity via flow cytometry analysis. Data presented were representative of three independent experiments (**p < 0.01, identified by one-way ANOVA with Dunnett’s multiple comparisons test). c, d AGS cells were treated with PPI in the absence or presence of GSH (4 mM) (c) or NAC (5 mM) (d) for 48 h in pH 7.4 condition. The expression level of SQSTM1 was measured by western blot. e, f AGS cells were exposed to various concentrations of PPI for 48 h in both pH 6.5 and pH 7.4 conditions. The levels of Nrf2 and its targets such as GCLC, GCLM, NQO1, and HO-1 were assessed by western blot analysis (e) and qRT-PCR analysis (f). Data were presented as mean ± SD (**p < 0.01, for each gene using one-way ANOVA Dunnett’s multiple comparison test). g–i AGS and HGC27 cells were reversely transfected with Nrf2 specific siRNA for 48 h, and then exposed to indicated concentrations of PPI for 48 h in both pH 6.5 and pH 7.4 conditions. Knockdown efficiency of Nrf2 was confirmed by western blot analysis (g, h), and the change pattern of SQSTM1 was evaluated by both western blot (g, h) and qRT-PCR analysis (i). Data were presented as mean ± SD (**p < 0.01, identified by two-way ANOVA with Tukey’s multiple comparisons test)