Cells were transfected with 0.5μg of AKT plasmid for 24h using Lipofectamine-LTX and treated with different concentrations of BITC for another 24h and analyzed by cell survival assay (A). Results were confirmed by three independent experiments. Control and BITC-treated AKT transfected cells were analyzed for p-AKT (Ser-473), AKT, p-FOXO1 (Ser-256), FOXO1, p-FOXO3a (Ser-253), FOXO3a, Bim, cleaved caspase-3 and cleaved PARP (B). Same blots were stripped and reprobed for β-actin. Cells from above mentioned treatment were also analyzed for apoptosis using annexinV by Accuri flow-cytometer (C). Each experiment was performed in triplicate. Results are expressed as Means±SD. (D) Effect of BITC with LY-294002. BxPC-3 cells were treated with LY-294002 and then with BITC (10μM, 24h). Cell lysates were analyzed by western blot.