Figure 2.

Combination of Olaparib and crizotinib induces autophagy in ovarian cancer cells. A and B, Analysis of LC3 and p62 protein level. Cells were treated with olaparib (olap, 5 µmol/L) and/or crizotinib (criz, 1 µmol/L). Cell lysates were collected, and Western blots were performed to examine the conversion of LC3-I to LC3-II and the p62 expression. The numbers below the gel lanes represent the relative protein levels determined from band intensities. Ratios of LC3-II/LC3-I and SQSTM1/p62 to GAPDH were calculated using ImageJ and normalized to the control. C, Analysis of autophagy by immunofluorescence staining of punctate LC3. Cells were plated in chamber slides and treated with olaparib (5 µmol/L) and crizotinib (1 µmol/L) for 48 hours. Representative images are presented here. Scale bar: 20 µm. Green dots indicate LC3 and (blue; DAPI) indicate nuclear staining. D, The average of LC3 puncta per cell was calculated by analyzing >100 cells per condition using ImageJ. Statistical analysis was conducted using one-way ANOVA. Error bars represent mean ± SD. E, Measurement of olaparib/crizotinib-induced autophagy with transmission electron microscopy (TEM) in OVCAR8, A2780 and OC316 cells. Red arrows indicate typical membrane autophagosomes.