Figure 1

AKT overexpression attenuates the effect of Par-4. (a) Effect of WA treatment on cell viability of DU-145, and DU-145/AKT cells for 24 h. The control cells were treated with DMSO or with the indicated concentration of WA for 24 h. Bars represents mean of three experiments with S.E. (b) PC-3 cells were transiently transfected with myr-AKT and empty vector. After transfection, cells were treated with or without WA 2 μ m. After 24 h, cells were harvested and cell lysates were prepared. Total cellular lysates were subjected to western blot analysis using antibodies against pAKT, AKT, and Par-4 proteins. β-Actin was used as a loading control. (c) DU-145/CMV and DU-145/AKT cells were treated with or without WA at a concentration of 2 μm concentration. Total cellular lysates were prepared and subjected to western blot analysis using antibodies against pAKT, AKT, and Par-4 proteins. β-Actin was used as a loading control. (d) RT-PCR showing Par-4 mRNA levels with WA treatment in PC-3 cells transfected with or without myr-AKT. (e) DU-145 and DU-145/AKT cells were treated with WA for 12 and 24 h, and RNA was isolated and subjected to RT-PCR analysis. (f) PC-3 cells were cotransfected with Par-4 promoter luciferase reporter construct, myr-AKT expression plasmid construct with renilla CMV as transfection control, and/or treated with WA. After 24 h, cells were harvested and assayed for luciferase reporter activity. Significant difference from control values was indicated at P<0.05 (Student's T-test). *P<0.05 and ^#P=0.06