Figure 6. CP-B2RAs induce apoptotic death and cell cycle arrest of MDA-MB-231 cells.

(A) Effects of B2RAs on apoptosis induction were investigated by Annexin V-FITC/ propidium iodide (PI) double staining followed by flow cytometry. Confluent cells were treated with the B2RAs at the indicated concentrations for 24h. Each bar represents mean values acquired from two independent experiments. (B) Effects of B2RAs on cellular senescence were analyzed by β-galactosidase staining following a 72h treatment. The pictures show representative contrast phase images of senescence-associated β-galactosidase expressing cells at 10x magnification. Serum-starved porcine cerebral microvascular endothelial cells (pCMVEC) served as a positive control for senescence. A repetition of the experiment gave similar results. (C) Effects of B2RAs on cell cycle were evaluated by flow cytometry of cells stained with PI. Each bar represents mean values acquired from two independent experiments. (D) Changed expression levels of cell cycle and apoptosis related proteins were evaluated after a 24h-treatment with B2RAs using Western blot analysis. Whole-cell lysates were blotted for the indicated proteins with the appropriate antibodies. Representative western blot of two independent experiments performed.