Fig. 1.
BNF inhibits cell proliferation, induces cell cycle arrest and cell senescence, and regulates the expression of cell cycle proteins MCF-7 cells. (A) 5×103 MCF-7 and MDA-MB-231 cells were seeded in 96-well plates. After an overnight culture, cells were treated with 10 µM BNF for 0, 1, 2 or 3 days. Cell proliferation was assessed by MTT. (B) Distribution of cell cycle in MCF-7 and MDA-MB-231 cells following treatment with 10 µM BNF for 48h. Upper panel, representative cell cycle distribution; Lower panel, quantitation of cell cycle distribution. (C) Expression levels of cell cycle proteins were detected by western blot in 10 µM BNF-treated MCF-7 and MDA-MB-231 cells for 48h. (D) The transcription level of p21Cip1/Waf1 was tested by qPCR in 10 µM BNF-treated MCF-7 and MDA-MB-231 cells. (E and F) BNF regulates the expression of cell cycle proteins by dose dependence (E) and time dependence (F) in MCF-7 cells. (G) Cell senescence was examined by SAβ-gal activity analysis. Right panel, representative senescent morphology; Left panel, quantitation of SAβ-gal activity, and SAβ-gal-positive rate (SAβ-gal+) (%) is calculated as follows: (number of SAβ-gal staining-positive cells/number of total cells) × 100. The data represent the mean ± SEM from three to five separate experiments. *P < 0.05, **P < 0.01 and ***P < 0.001, versus DMSO-treated cells.