(A), Inhibition of FOXO transcription factor by shRNA blocks anti-proliferative effects of resveratrol. LNCaP cells were transiently transfected with plasmids expressing FKHR shRNA, FKHRL1 shRNA, AFX shRNA or respective scrambled control and treated with resveratrol (20 µM) for 48 h, and cell viability was measured. (B), Inhibition of FOXO transcription factors or ROS (reactive oxygen species) by NAC blocks resveratrol-induced apoptosis. LNCaP cells were transfected with a mixture of plasmids expressing FKHR shRNA, FKHRL1 shRNA plus AFX shRNA or scrambled control. After transfection, the culture medium was changed and cells were pretreated with NAC for 2 h, and treated with resveratrol (20 µM) for 48 h. At the end of incubation period, the apoptosis was measured by TUNEL assay. (C), Inhibition of FOXO transcription factors or ROS by NAC blocks resveratrol-induced caspase-3 activity. LNCaP cells were transfected with a mixture of plasmids expressing FKHR shRNA, FKHRL1 shRNA plus AFX shRNA or scrambled control. After transfection, the culture medium was changed and cells were pretreated with NAC for 2 h, and treated with resveratrol (20 µM) for 48 h. At the end of incubation period, the caspase-3 activity was measured as per manufacturer instructions.