Fig. 6

XPO1 inhibitor KPT-330 activated p53/mTOR pathway to induce autophagy-dependent apoptosis. A Expression of LC3- II/I, Cleaved PARP, Cleaved caspase 3 was detected by western blot after KPT-330 or Z-VAD-FMK treatment. Cells were pre-treated with Z-VAD-FMK (concentration of 0.1 μM) for 6 h, then were treated with KPT-330 for 48 h. B Expression of LC3- II/I, Cleaved PARP, Cleaved caspase 3 was detected by western blot after KPT-330 or chloroquine treatment. Cells were pre-treated with chloroquine (concentration of 0.2 μM) for 6 h, then were treated with KPT-330 for 48 h. C Western blot analysis of p53 levels in nucleus and cytoplasm of NOZ and GBC-SD showed that more p53 proteins were accumulated in nucleus after KPT-330 treatment. D Immunofluorescence images of p53 showed that more p53 proteins were accumulated in nucleus in NOZ and GBC-SD after KPT-330 treatment. Scale bars represent 75 μm. E P21 and P27 expression levels were detected by western blot after KPT-330 treatment for 48 h. F Expression of LC3- II/I, p53, p-mTOR/mTOR was detected by western blot after KPT-330 or p53-siRNA treatment. Cells were pre-treated with p53-siRNA for 48 h, then were treated with KPT-330 for 48 h. G Expression of LC3- II/I, p53, p-mTOR/mTOR, Cleaved PARP and Cleaved caspase 3 was detected by western blot. GBC cells were pre-treated with p53-siRNA for 48 h or MHY1485 (concentration of 0.5 μM) or chloroquine (concentration of 0.2 μM) for 6 h, then were treated with or without KPT-330 for 48 h. H Knockdown of p53 attenuated inhibition effects of KPT-330 in NOZ and GBC-SD assessed by cell proliferation assays. GBC cells were pre-treated with p53-siRNA for 48 h, then were treated with or without KPT-330 for 48 h. Student's t test was applied to the statistical analysis in this figure. Data presented as mean ± SD (n = 3)