Figure 5

Apoptotic cell-death measured by flow cytometer in breast CSCs. (a) Breast CSCs were treated with Rott (0, 0.5, 1 and 2 μM) in complete stem cell culture medium for 48 h and apoptosis was measured by PI staining followed by flow cytometry. Data are the means of triplicate experiments. (b) Time-course evaluation of spontaneous apoptosis of breast CSCs treated with Rott (0, 0.5, 1 and 2 μM) in complete stem cell culture medium for 48 h and apoptosis was measured by annexin-V/PI staining followed by flow cytometry. Data are the means of triplicate experiments. Representative histograms are shown of Rott treated breast CSCs stained with annexin-V and propidium iodide. After 48 h of culture, three populations of cells were observed: viable cells (negatively stained, lower left quadrants); early apoptotic cells (annexin-V positive and propidium iodide negative, lower right quadrant) and cells in the late stages of apoptosis (annexin-V and propidium iodide positive, upper right quadrants). By increasing Rott concentration at 48 h, a greater number of breast CSCs underwent the early and late stages of apoptosis. (c) Kinetic evaluation of cells undergoing the early stage of apoptosis in breast CSCs. Cells staining with annexin-V only, showing the early stage of apoptosis (% early apoptosis) (d) Kinetic evaluation of cells underwent the late stage of apoptosis in breast CSCs. Cells staining with annexin-V alone and with both annexin-V and propidium iodide were combined, giving the total number of cells at both the early and late stages of apoptosis (% cell death). (e) Kinetic evaluation of cells underwent apoptotic cell death in breast CSCs. Data are reported as the mean ± standard error (SE) of percentage of cells. n = 5, *P < 0.05 when compared with Rott treated in an identical manner.