Figure 2

APE induces apoptosis in MDA-MB-231 cells. (a) MDA-MB-231 cells were treated with APE 100 and 300 μM EqC for 24 h. Flow cytometry analysis was then performed. Representative dot plots of both Annexin V-FITC and PI-stained cells. The percentage of cells is reported in the quadrants. Viable cells, lower left; non-viable necrotic cells, upper left; early apoptotic cells, lower right; late apoptotic cells, upper right. For each sample 2 × 10⁴ events were acquired. Analysis was carried out by triplicate determination on at least three separate experiments. (b) MDA-MB-231 cells were incubated with APE 100 and 300 μM EqC for 24 h. Cell lysates were prepared and analyzed by western blotting assay for the expression of apoptosis-related proteins. The graph shows the densitometric intensity of Bax/Bcl-2 ratio. The intensities of signals were expressed as arbitrary units. The house-keeping protein β-actin was used as loading control. (c) MDA-MB-231 cells were treated with APE 300 μM EqC for 24 h with or without co-treatment with 40 µM Z-IETD-FMK caspase-8 inhibitor and cell apoptosis was evaluated by flow cytometry. (d) The level of procaspase-3 in MDA-MB-231 cells treated with APE 300 μM EqC for 24 h with or without Z-IETD-FMK was measured by western blotting. The graph shows the densitometric intensity of caspase 3 band under the different experimental conditions. All results were obtained from at least three independent experiments (*P < 0.05 versus control).The full-length blots are included in the supplementary information (Figs S2, S3).