Fig. 2

β-escin-induced apoptosis is associated with activation of caspases and dysregulation of mitochondrial proteins. A, B The effects of β-escin (10-30 μM, 48 h) on apoptotic-related proteins. BT474 and JIMT-1 cells were treated with β-escin (10-20 μM and 20-30 μM, respectively) for 48 h. The expression of cleaved-caspase-7, cleaved-caspase-3, and cleaved-PARP were upregulated in both BT474 (A) and JIMT-1 cells (B). Quantitative graphs of protein content are shown in the right panels (*p<0.05). C, D The effects of β-escin (10-30 μM, 48 h) on the expression of Bcl-2 family proteins including Bcl-2 and Bax in BT474 (C) and JIMT-1 cells (D). Quantitative graphs represent the ratio of Bcl-2/GAPDH and cleaved-Bax/GAPDH (right panels, **p<0.01). Results are shown as mean ± SEM of at least three independent experiments. Data were analyzed by one-way ANOVA and Bonferroni’s post hoc test. E β-escin increases ROS accumulation. BT474 and JIMT-1 cells were treated with β-escin (20 or 30 µM, respectively) for 3 and 6 h. The cells were stained with DCF-DA and analyzed by flow cytometry. F β-escin induces release of cytochrome c. JIMT-1 cells were treated with β-escin at 30 µM for 24 h and immunostained for cytochrome c (green) and TOM 20 (red) with DAPI (nuclei, blue). The cytochrome c intensity (green line) and cellular localization (yellow or green arrows) were analyzed with confocal microscopy using the intensity profile tool (straight yellow line). The mean fluorescence intensity (MFI) representing cytosolic cytochrome c were measured using the histogram tool (****p<0.0001, right panel). Cyto c, cytochrome c; TOM 20, translocase of outer mitochondrial membrane 20