Figure 2

Effects of GFW on cell cycle and induction of apoptosis in mouse urothelial cell line, MB49. (A) MB49 cells were treated with GFW at concentrations of 0.5, 1 and 2 mg/mL for 24 h and then stained with propidium iodide and analyzed using flow cytometry. Data are represented as mean ± SEM (n = 4). Significant differences from the control are indicated by *p < 0.05, **p < 0.01 or ***p < 0.001, as determined by one-way ANOVA and Dunnett comparison test. (B) Analysis of apoptosis using flow cytometry. MB49 cells were treated with GFW at concentrations of 0.5, 1 and 2 mg/mL for 24 h, stained with propidium iodide and Annexin V and then analyzed using flow cytometry. The lower left quadrant (Q3) represents viable cells, the upper left quadrant (Q1) represents necrotic cells, the lower right quadrant (Q4) represents early apoptotic cells and the upper right quadrant (Q2) represents nonviable late apoptotic cells. The relative percentages include early and late apoptotic cells. Data are represented as mean ± SEM of three independent experiments. Significant differences from the untreated control are indicated by *p < 0.05, **p < 0.01 or ***p < 0.001, as determined by one-way ANOVA and Dunnett comparison test. (C) Analysis of caspase-3 activation in GFW-treated cells. Western blot analysis was performed to detect caspase-3 and its cleaved form in MB49 cells after treatment with 2 mg/mL GFW for various durations. β-Actin protein was used as the loading control.