Figure 5.

Inhibition of olaparib/crizotinib-induced ROS increases cell growth, decreases apoptosis and inhibits autophagy. A, Assessment of the effect of ROS inhibition on induction of autophagy. OVCAR8, A2780 and OC316 cells were treated with olaparib/crizotinib for a total of 48 hours and NAC (1 mmol/L) was added to culture media 24 hours before analysis. Western blot analysis was performed to evaluate conversion of LC3I/LC3II. B, Measurement of cell viability with and without NAC treatment. Cells were treated with olaparib, crizotinib and/or NAC for 5 days. After incubation, cells were fixed and stained by sulforhodamine B (SRB). C, Analysis of apoptosis with and without NAC treatment. Cells were labeled with PI/annexin V-FITC and analyzed for apoptosis by flow cytometry. Data represents results from two independent experiments with three replicates. Results were obtained using one-way ANOVA. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. D, Evaluation of the expression of AKT/mTOR-ULK1 after treatment with olaparib, crizotinib, NAC or combination for 48 hours, NAC was added 24 hours before analysis. Numbers below the gel lanes represent the relative protein level, determined from the band intensity. GAPDH for (D) from OC316 cells is the same as that presented for the same cells in (A) since the blots in each panel were performed using the same membranes.