Figure 5

Combining BEZ235 with CI-1040-induced BIM expression and sensitizes SKOV3 cell line to miR-491-5p. (a) SKOV3 cells were transfected with miR-491-5p or miR-Ctrl for 72 h. Whole-cell extracts were analysed by western blotting with the indicated total and phospho-specific antibodies. β-actin level is shown as loading control. (b) SKOV3 cells were treated with DMSO or 5 μM CI-1040 (MEK inhibitor) or 1 μM BEZ235 (a dual PI3K and mTOR inhibitor) or with both pharmacological inhibitors (BEZ235 and CI-1040) for 72 h. Whole-cell lysates were then subjected to western blot analysis with the indicated total and phospho-specific antibodies. β-actin level is shown as loading control. (c and d) SKOV3 cells were transfected with miR-491-5p or miR-Ctrl followed by treatment with DMSO or with BEZ235 or CI-1040 or both pharmacological inhibitors (BEZ235+CI-1040) for 72 h. (c) Representative cell morphology and DNA content histograms obtained by flow cytometry are shown. The percentage of cells in sub-G1 phase was indicated for each condition. Similar results were obtained in three independent experiments. (d) Protein lysates were immunobloted with the indicated total and phospho-specific antibodies. β-actin level is shown as loading control. (e) SKOV3 cells were co-transfected with miR-491-5p and siRNA against BIM (siRNA-BIM) or siRNA control followed by a treatment with BEZ235+CI-1040 for 72 h. Whole-cell lysates were analysed by western blotting for PARP (native and cleaved forms) and caspase-3 (pro and cleaved (active) forms)