Figure 4

ATF2 affects the PTX-elicited activation of Bim and Puma. (A) B16F10 cells treated with PTX were examined for the subcellular localization of proteins by western blotting, with the protein weights indicated (in kDa). COXIV was shown as a loading control of mitochondrial sample; β-Actin was shown as an loading control of cytosolic fraction. B16F10 cells stably transfected with scrambled or Bim SiRNA (B), VDAC1 SiRNA (C) and ATF2 shRNA (D) were subjected PTX treatment and then subject to immunoblot analysis with the indicated antibodies. B16F10 cells were preincubated with or without DIDS (100 μM, for 1.5 h), followed by ABT737 (1μM, 24 h) and PTX treatment (100 nM, 12 h). Cells were subjected to immunoblot analysis with the indicated antibodies (E) and FACS apoptosis analyses Annexin V/PI staining (F), *P < 0.01, ANOVA. Data are means ± s.e.m. (n = 5). (G) B16F10 cells stably transfected with scrambled or ATF2 shRNA were incubated with ABT-737 alone or in conjunction with PTX. The cells were immunofluorescently stained with the indicated antibodies and visualized using confocal microscopy (Olympus 1X81) (Scale bar: 20 μm).