Figure 2

Salinomycin inhibits cell growth and induces apoptosis through cell cycle arrest at G1 phase. (a) Cells were treated with indicated concentrations of salinomycin for 72 h, and cell growth inhibition was determined by MTS assay. Data are presented as averages of triplicate measurements, with error bars representing standard deviations. *P<0.05, **P<0.005, and ***P<0.0005. (b) Cells were treated with 4 μM salinomycin or DMSO vehicle, and cell numbers were counted using a cell counter at the indicated time points. (c) Cells were treated without or with 4 μM salinomycin for 72 h, and then apoptosis was measured by flow cytometric analysis of cells stained with annexin V and PI. The percentages of annexin V- or/and PI-positive cells are indicated. Data are presented as averages of triplicate measurements, with error bars representing standard deviations. *P<0.05, **P<0.005, and ***P<0.0005. (d) Cells were treated without or with 4 μM salinomycin for 72 h. Cells were then replated and incubated in drug-free media for 10 days to allow colony formation. Colonies were stained with crystal violet. (e) Cells were treated without or with 4 μM salinomycin for 72 h, and then equal amounts of cell lysates were subjected to immunoblot analysis using the indicated antibodies. Similar results were observed in independent experiments. Arrows indicate cleaved forms of each protein. (f) Cells were treated without or with 4 and 8 μM salinomycin for 24 h, and images were taken using phase contrast microscopy. Magnification: × 50; scale bar=100 μm. (g) Cells were treated without or with 4 μM salinomycin for 24 h, and then cell cycle was analyzed by flow cytometry. The percentage of cells in G1 phase is annotated in each column. Similar results were observed in three independent experiments