Figure 2

Bim and VDAC1 are involved in ATF2-associated apoptotic induction. (A) Different cancer cell lines were subjected to mitochondria-mediated apoptosis with PTX, STS (1.25 μM, 5 hours), and cisplatin (50 μM, 30 hours) and apoptosis analysis by Annexin V/PI staining. Data are means ± s.e.m and represent three different experiments. *P < 0.01, ANOVA, versus Control. (B) The B16F10 cells were transfected with siBim (low panel), shATF2 (middle panel) and ATF2^T52A (up panel), and whole-cell lysate for siBim and shATF2 or fractionated to Nuclear and mitochondrial fractions for ATF2^T52A were subject to immunoblot analysis with indicated antibodies. β-actin loaded as control. The morphometry of ATF2 are indicated at right. The columns provide average values of at least 3 independent experiments performed in triplicate; the bars, SE. *P < 0.01 compared with the corresponding control. (C) B16F10 cells, transfected with ATF2^T52A or not were treated with PTX (100 nM, 12 hours), pretreatnent with or without DIDS (100 μM, 1 h). Cells were then subjected to measurement of apoptosis by Annexin V/PI staining. Columns, mean of three individual experiments; bars, s.e.m. *P < 0.01, ANOVA. (D) Cells expressing shATF2 were exposed in the absence or presence of ABT-737 (1μM, 24 h) and subjected to mitochondria-mediated apoptosis induction by PTX. Cells were then subjected to measurement of apoptosis by Annexin V/PI staining. Columns, mean of three individual experiments; bars, s.e.m. *P < 0.01, ANOVA. (E) Cells expressing siBim were exposed in the absence or presence of ABT-737 and then subjected to mitochondria-mediated apoptosis induction by PTX. FACS analyses of apoptotic cell death were performed using Annexin V/PI staining. Columns, average values of three independent experiments; bars, s.e.m. *P < 0.01, ANOVA.