Fig 4. The combination of dual PI3K and AKT inhibitors regulate colony formation and apoptosis.

(A) RT112 cells were treated with control (ctrl), 1000 nM MK-2206, 500 nM GSK2334470, 200 nM NVP-BEZ235 or their combinations for 12 days and colonies were stained with 0.5% crystal violet. (B) Quantification of surviving colonies in duplicate samples relative to control expressed as a percentage and three independent experiments. (C) Cells were lysed and immunoblotting was performed with the indicated antibodies after treating cells with the respective inhibitors or control (ctrl) for the indicated time points. Results are representative of at least three independent experiments. (D) RT112, T24 or 647V cells were treated with 200 nM, 100 nM and 200 nM NVP-BEZ235 respectively, 1000 nM MK-2206, their combination or control (ctrl) for 48 hours and caspase 3/7 assay was performed. Results obtained in arbitrary luminescence units were normalized to the number of living cells per trypan blue staining conducted in parallel and indicate the mean +/- standard error of three independent experiments relative to control expressed as a percentage. * indicates p < 0.05 (unpaired Student’s T-test.