FIGURE 4.

The inhibitory effects of safranal on AKT phosphorylation and transcriptional activity of E2F1 during quiescent Pca cell re-entry. Quiescent LNCaP (A) and PC-3 (B) cells were initially stimulated to re-enter the cell cycle, and the effects of safranal (GI₉₀) on protein expression levels of p-AKT (Ser473), AKT, and E2F1 at specific intervals were determined by immunoblotting. GAPDH served as a loading control. The mRNA expression levels of E2F1 in LNCaP (C) and PC-3 (D) cells during cell cycle re-entry in the presence or absence of safranal (GI₉₀) were examined by RT-qPCR. E2F1 levels in cell nuclear and cytoplasm extracts of LNCaP (E) and PC-3 cells (F) were analyzed using immunoblotting after treatment with safranal for 12 and 3 h, respectively, following release from the quiescent state. α-Tubulin and lamin A/C served as loading and purity controls for the cytoplasm and nuclear fractions, respectively. Following a 6-day serum withdrawal for LNCaP and 2-day contact inhibition for PC-3 cells, quiescent LNCaP (G) and PC-3 (H) were transfected with pGM-E2F-Luc and renilla luciferase reporter plasmid pML-SV40-hRluc by using the EZ transfection agent for 15 h. The transfected quiescent cells were then induced to re-enter the cell cycle in the presence or absence of safranal (GI₉₀) for 24 h. Cell lysates were collected to assess firefly and renilla luciferase activities using the dual luciferase reporter assay kit. Renilla luciferase served to normalize the values of the experimental reporter gene and acted as an internal control for transfection efficiency. Data are expressed as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 vs. dimethyl sulfoxide vehicle control.