Effect of Glibenclamide on cell cycle progression. Panel A: Synchronized MDA-MB-231 cells were treated with IC50 Gli (25 μM) or vehicle for 24, 48 or 72 h and the fraction of cells in each phase of cell cycle was evaluated by flow citometry. Gli treatment clearly arrested cells at G0/G1 phase. Results are expressed as percentage of the value obtained with vehicle (means ± SEM of three experiments on parallel). °p < 0.01 vs control; *p < 0.001 vs control, t test. Left bars: control; right bars: Gli-treated cells. Panel B: A decrease in BrdU incorporation to DNA was observed when cells were treated with 25 μM Gli for 48 h. Results are expressed as the means ± SEM of three experiments on parallel. *p < 0.05 vs. control, t test. Panel C: Expression of G1-S regulatory proteins in MDA-MB-231 cells treated with Gli or vehicle for 72 h was analyzed by Western blot. Gli decreased the level of cyclin E and increased p27Kip1. Representative immunoblot images of cyclins D1, B1, E and p27Kip1 are illustrated. Relative quantification was performed by densitometric analyses. Actin densitometric values were used to standardize for protein loading. Bars represent the mean ± SEM of three independent experiments. **p < 0.01 vs control; ***p < 0.001 vs control, t test.