Nobiletin inhibits VEGF expression by regulating AKT and HIF-1α gene in PC-3 cells. (a) Nobiletin inhibits VEGF expression by regulating HIF-1 α gene, (b) Nobiletin inhibits VEGF expression by regulating AKT gene. PC-3 prostate cancer cells were seeded in 96-well plate at 10,000 cells/well and incubated overnight. The cells were then transfected with 0.05 ug VEGF (Hif-1α) luciferase reporter, 0-0.25 μg HIF-1α (AKT) or SR-α plasmids by 0.6 μL jetPRIME reagent for 4 hours, followed by 16 hour treatment with 0 or 40 μM nobiletin. The cells were harvested and analyzed for luciferase and total protein levels, and the levels of VEGF (Hif-1α) reporter were normalized by corresponding total protein levels. Data represent Means ± SE from 3 independent experiments. #p < 0.05 as compared to control. ##p <0.01 as compared to control. *p <0.05 as compared to nobiletin-treated control. **p <0.01 as compared to nobiletin-treated control.