Fig. (4).

PD downregulates MDM2 and increases FOXO3a expression in PC3 cells. A. PC3, LNCaP and DU145 cells were exposed to various concentrations of PD for 24 hr; then target proteins (MDM2, FOXO3a, p53, p21and p27) were examined by Western blotting (WB). B. The expression of the FOXO3a and p-FOXO3a proteins was examined by WB in PC3 cells after PD treatment for 24 hr. C. & D. PC3 cells were transfected with MDM2 siRNA or a pcDNA-MDM2 (full length) plasmid,then the expression levels of MDM2 and FOXO3a were determined by WB after incubation with or without 10 mM PD. Blocking MDM2 expression by siRNA increased the FOXO3a expression, while MDM2 overexpression decreased the FOXO3a protein expression. However, treatment with PD was able to increase the FOXO3a expression even in the presence of MDM2 overexpression. E. The mRNA expression levels of MDM2, FOXO3a, p21 and p27 were detected by real-time quantitative PCR, and the data were normalized to the mRNA level of GAPDH.