Quercetin induced Foxo3a activity.
(A) MDA-MB-231 cells were transiently transfected with Foxo3a luciferase reporter for 24 h prior to treatment of 20 µM quercetin for another 24 h. Thereafter, cell lysates were collected for Luciferase assay. The graph shows relative luciferase activity. (B) RT real-time PCR analysis for expression of Foxo3a mRNA in MDA-MB-231 cells treated with 20 µM quercetin for 12 h, 24 h and 48 h. (C) MDA-MB-231 cells were treated with 20 µM quercetin for various time points from 0 to 48 h and total Foxo3a was detected by western blot. β-actin was used as loading control (left). Densitometric quantification of Foxo3a protein levels from western blot analysis (right). Foxo3a levels were compared to basal level at 0 h. (D) After treatment with quercetin for 24 h, cells were fixed and stained for Foxo3a, and DNA was counterstained with DAPI. Representative microcopy images are shown (E) Breast cancer cells were treated with quercetin for 6 h, 12 h and 24 h, and subcellular fractions were isolated and immunoblotted for Foxo3a. Lamin B1 and β-actin were used as loading controls of nucleus and cytoplasm respectively. Western blot analysis image (up) and densitometric quantification of Foxo3a protein expression from western blot analysis (down). *p<0.05, **p<0.01, ***p<0.001.
