FIGURE 5.

The inhibitory effect of safranal on the activation of the canonical and non-canonical NF-κB pathways during quiescent Pca cell re-entry. Following the induction of quiescent LNCaP (A) and PC-3 (B) cell re-entry into the cell cycle, the effects of safranal (GI₉₀) on the protein expression levels of p-IKKα/β (Ser176/180), IKKα/β, p-IκBα (Ser32), IκBα, NF-κB p65, and p100/p52 at the indicated intervals were determined by immunoblotting. GAPDH served as a loading control. The mRNA expression levels of p65 and p52 in LNCaP (C) and PC-3 (D) cells during cell cycle re-entry in the presence or absence of safranal (GI₉₀) were assessed by RT-qPCR. NF-κB p65 and p52 levels in cell nuclear and cytoplasm extracts of LNCaP (E) and PC-3 cells (F) were analyzed by immunoblotting after treatment with safranal for 24 and 1 h, respectively, following release from the quiescent state. α-Tubulin and lamin A/C were used as loading and purity controls for the cytoplasm and nuclear fractions, respectively. Following a 6-day serum withdrawal for LNCaP (G) and a 2-day contact inhibition for PC-3 cells (H), the cells were transfected with pML-NFκB-Fluc2-Neo enhanced 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 determine 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 as an internal control for transfection efficiency. Data are expressed as mean ± SD. ***P < 0.001 vs. dimethyl sulfoxide vehicle control.