Fig. 7. Bcl-2 inhibitors synergized the cytotoxicity of PPI.

a, b The Bcl-2 family members were analyzed by western blot (a) and qRT-PCR (b). Data were presented as mean ± SD (**p < 0.01, for each gene using one-way ANOVA with Dunnett’s multiple comparisons test). c AGS cells were pretreated with various concentrations of PPI for 24 h in pH 7.4 condition, and then incubated with two different doses of ABT-263/ABT-737 for another 24 h. Data were presented as percentages of cell viability as determined by CCK8 assays (upper panel). Synergisms of cell viability inhibition by the combination therapy were analyzed by Combination Index Value (lower panel). d Cell viability of AGS cells after treatment with combination of 100 μg/ml PPI and 4 μM ABT-263/10 μM ABT-737 in pH 7.4 condition. The combination indexes (CI) were calculated as described in Methods section. Data were presented as mean ± SD (***p < 0.001, indentified by one-way ANOVA Dunnett’s multiple comparison test). e AGS cells were treated as described in (c), and the changes in mitochondrial membrane potential (Δψm) were analyzed by JC-1 assay. Ratio of green to red fluorescence was depicted (left panel). Data were representative of three independent experiments. Right panel showed the quantification of the Δψm in AGS cells upon combinational treatment. Data were presented as mean ± SD (***p < 0.001, indentified by one-way ANOVA Tukey’s multiple comparisons test). f AGS cells were treated as described in (c), and then stained with MitoTracker green (100 nM) and MitoTracker red (400 nM). The dysfunctional mitochondria accumulation was analyzed by FACS. Dot plots of subpopulation are depicted (left panel) and percent of dysfunctional mitochondria were shown as mean ± SD (right panel) (***p < 0.001, indentified by one-way ANOVA Tukey’s multiple comparisons test). g AGS cells were treated as described in (c). The apoptosis related proteins were detected