Figure 7. Effect of Akt inhibition on BA145 induced autophagy and G2/M arrest.

(a) Pharmacological inhibition of Akt augments BA145 induced autophagy. PANC-1 cells were treated with BA145 (40 μM) and/or specific Akt inhibitors Akt inhibitor IV (5 μM) and Akt inhibitor VIII (2 μM) for 12 h time period. Cells were stained with acridine orange (1 μg/ml) for 10 min, and analyzed by fluorescent microscopy. Cotreatment of BA145 with Akt inhibitors enhanced the accumulation of red coloured acidic vesicles in PANC-1 cells (b) Effect of Akt inhibitors on cell cycle progression in BA145 treated PANC-1 cells. Cells were treated with BA145 and/or Akt inhibitors at above described concentrations for 12 h time period. Cells were stained with PI and cell cycle populations were analyzed by flow cytometry. Pharmacological inhibition of Akt enhanced cell population in G2/M phase in BA145 treated PANC-1 cells (c) Akt inhibitors augmented the inhibitory effect of BA145 on cell cycle regulatory proteins in PANC-1 cells. After BA145 treatment with/or Akt inhibitors, cells were lysed with lysis buffer and protein lysates were prepared for western blotting of indicated proteins. β-actin was used as loading control (d) Genetic silencing of Akt through gene specific siRNA enhanced the accumulation acridine orange positive acidic vesicular organelles in BA145 treated PANC-1 cells (e) Influence of Akt inhibition by siRNA in the expression of G2/M regulatory proteins in BA145 treated PANC-1 cells. BA145 has more inhibitory effect on the expression of cell cycle regulatory proteins in Akt silenced cells as compared to scrambled siRNA control cells.