Figure 4

RAP1 desensitizes NSCLC cells to CP-induced cell death. (a) shRAP1- or scramble shRNA-transduced A549 cells were treated with different concentrations of CP for 48 h, and cell viability was determined with CCK-8 assay and normalized to the same type of cells cultured without CP. (b) Proliferation of A549 cells after transduction with control (CTRL) or RAP1-overexpressing (RAP1) lentiviral vectors. (c) Cell viability of A549 cells transduced with RAP1 or CTRL viruses and treated with different concentrations of CP for 48 h, after normalization to the same type of cells cultured in CP-free media. (d) Competition assay of the untransduced (GFP⁻) cells co-cultured with the cells transduced with the indicated different RAP1 modification lentiviruses (GFP⁺). Cells were treated with 2 μM of CP and harvested at 24 and 72 h. Percentage of GFP⁺ cells were analyzed for the viable population determined by 4,6-diamidino-2-phenylindole staining. (e and f) Untransduced (UnTxD) A549 cells and A549 cells with RAP1 overexpression (RAP1) or RAP1 knockdown (shRAP1) were cultured in media containing 1 μM CP (+) or CP-free media (−) for 24 h and analyzed for the apoptotic status with western blotting analysis for cleaved caspase-3 (C-Cas3) and BCL-2 (e) or with flow cytometric analysis of Annexin-V (f), representatives of three independent experiments. Statistics were generated from three independent experiments. *P<0.05, **P<0.01, ***P<0.001, Student’s t-test; error bar: ±S.D.