Effects of AKT3 expression on apoptosis of PCa cell lines were analyzed by Annexin V/propidium iodide (PI) staining and caspase 3/7 activity. Cells were transfected with control or AKT3 expression plasmid and apoptosis was induced by adding FasL (200 ng/mL), enzalutamide (10 µM) or docetaxel (20 nM) 24 h after transfection. Staining with Annexin V-FITC/ PI was performed 72 h post treatment and followed by flow cytometry analysis. Graphs show the mean ± SEM of four independent experiments. (A) Percentage of apoptotic cells decreased significantly (*** p < 0.001) after AKT3 overexpression in untreated LNCaP cells (p = 0.0008) as well as FasL (p = 0.0001) and enzalutamide (p = 0.0002) treated LNCaP cells. (B) Caspase 3/7 activity was measured 48 h after treatment by microplate reader. AKT3 overexpression in LNCaP cells has a slightly effect on caspase 3/7 activity. After induction of apoptosis with FasL (p = 0.0377; * p < 0.05) or enzalutamide (p = 0.0004; *** p < 0.001), AKT3 overexpression leads to a significant reduction of caspase 3/7 activity in LNCaP cells. AKT3 expression in 22Rv1, DU145 and PC3 cells has no effect on the relative proportion of apoptotic cells (C,E,G) as well as on caspase 3/7 activity (D,F,H).