Fig. 4

ER stress is involved in (S)-crizotinib-induced NSCLC cell apoptosis. a NCI-H460 cells were treated with 30 μM (S)-crizotinib for the indicated times and protein levels of ATF4, phosphorylated eIF2α, and CHOP were determined by western blot. GAPDH was used as loading control. b NCI-H460 and H1975 cells were treated with (S)-crizotinib at different concentrations for 6 h. Cell lysates were then subjected to western blotting for ATF4 and p-eIF2a levels. c NCI-H460 and H1975 cells were pretreated with 5 mM NAC for 1 h before exposure to (S)-crizotinib for 6 h. ATF4, p-eIF2α levels were detected by western blot. d-e Levels of CHOP in NCI-H460 cells following treatment with 30 μM (S)-crizotinib for different time periods. The exposure to (S)-crizotinib was carried out with or without pretreatment of cells with 5 mM NAC for 1 h (e). f Effect of (S)-crizotinib on the morphology of endoplasmic reticulum in NCI-H460 cells. Cells were treated with 30 μM (S)-crizotinib before examination by an electron microscope (×10,000 or ×20,000). Presentative micrographs from three independent experiments are shown. g-h NCI-H460 cells were transfected with siRNA against CHOP or control siRNA. Cells were then treated with 30 μM (S)-crizotinib. GAPDH was used as internal control for western blot analysis (g). Relative cell viability was determined by counting viable cells under a microscope (h) [*P < 0.05 and **P < 0.01]