Figure 5

CP resistance is associated with RAP1-dependent NF-κB signaling induction. (a) Schema of stepwise CP treatment on A549 cells. (b and c) RAP1 expression and NF-κB activation during CP treatment. A549 cells treated with CP as depicted in panel (a) were harvested at the indicated time points; the protein expression of RAP1, p65, pp65 and p-IκBα in the cytoplasmic and nuclear fractions was measured with western blotting analyses (b), representatives of three independent experiments; and the mRNA expression of NF-κB signaling downstream factors was analyzed with qRT-PCR (c). (d) A549 cells with overexpressed RAP1 were treated with CP as depicted in panel a. RAP1, pp65 and p-IκBα were detected in the cytoplasmic (Cyto) and nuclear (Nu) fractions of cell lysate at the indicated time points, representatives of three independent experiments. (e) Cell viability of A549 cells transduced with shRAP1 or scramble shRNA at different time points during the sequential CP treatment as depicted in panel a, normalized to the same type of cells cultured in CP-free media in respective time point. (f) Nuclear (upper panel) and cytoplasmic (lower panel) fractions were isolated from A549 cells transduced with shRAP1 or scramble shRNA and treated with 0.5 μM of CP for 24 h and assayed for p65, pp65 and p-IκBα protein levels, representatives of three independent experiments. (g) mRNA expression of NF-κB signaling downstream factors in shRAP1-transduced A549 cells after 24 h culture in the presence or absence of 0.5 μM of CP. Statistics were generated from three independent experiments. **P<0.01, ***P<0.001, Student’s t-test; error bar: ±S.D.