Figure 6

RAP1 deletion is lethal to CP-resistant NSCLC cells. (a and b) Protein expression of RAP1, pp65, p-IκBα (a) and mRNA expression of NF-κB signaling downstream factors (b) in control (CTRL), RAP1-overexpressed (RAP1) and CP-resistant (A549R) cells, representatives of three independent experiments. (c) Cell proliferation of A549R cells transduced with scramble shRNA or shRAP1 assayed with CCK-8 staining at the time point of transduction. Cells were precultured in CP-free media for 24 h before transduction. (d) RAP1-deleted A549 cells and scramble shRNA-transduced A549R cells were cultured in media containing 1 μM CP (+) or CP-free media (−) for 24 h before being lysed to harvest protein. Lentiviral shRAP1 was introduced to A549R cells precultured in CP-free media for 24 h and protein harvested 12 h after transduction. Cleaved caspase-3 and BCL-2 were detected by western blotting. (e) IF assay of cleaved caspase-3 (red) in untreated A549 and A549R cells transduced with scramble shRNA or shRAP1. (f) Flow cytometric analysis for Annexin-V staining in A549 and A549R cells after RAP1 deletion. A549 cells were maintained in CP-free media and A549R cells were preconditioned in CP-free media for 24 h before analysis. Statistics were generated from three independent experiments. *P<0.05, **P<0.01, ***P<0.001, Student’s t-test; error bar: ±S.D.