CRISPR/Cas9 Knockout of Bak Mediates Bax Translocation to Mitochondria in response to TNFα/CHX-induced Apoptosis

Figure 2

BAK knockdown, but Bax, blocked TNFα/CHX-induced apoptosis. (a) Bak knockdown inhibited TNFα/CHX-induced apoptosis in HeLa cells. After transfection of cells with non-specific siRNA (lanes 1 and 2) and specific Bak siRNA (lanes 3 and 4), cells were subjected to co-treatment with TNFα/CHX (+) for 5 h or left untreated (−). Western blots using antibodies against Bak (panel 1); PARP (panel 2); caspase-9, -8, and-3 (panels 3–5); and β-actin (bottom panel) are shown (relative sample loading). (b) Bak knockdown inhibited TNFα/CHX-induced apoptosis in SH-SY5Y cells. After transfection of SH-SY5Y cells with non-specific siRNA (lanes 1 and 2) and specific Bak siRNA (lanes 3 and 4), cells were subjected to co-treatment with TNFα/CHX (+) for 5 h and 7 h or left untreated (−). Western blots using antibodies against Bak (panel 1), PARP (panel 2), and β-actin (panel 3) are shown (relative sample loading). (c) Bax knockdown had no effect on TNFα/CHX-induced apoptosis in HeLa cells. After transfection with non-specific siRNA (lanes 1 and 2) and specific Bax siRNA (lanes 3 and 4) into HeLa cells, cells were subjected to co-treatment with TNFα/CHX (−) for 5 h or left untreated (−). Western blots using antibodies against Bak (panel 1), PARP (panel 2), and β-actin (panel 3) (relative sample loading). (d, e) Bak knockdown inhibited TNFα/CHX-induced PARP cleavage in A549 and SW480 cells. After transfection with non-silencing siRNA (lanes 1 and 2) and specific Bak siRNA (lanes 3 and 4) into A549 (d) and SW480(e) cells, cells were subjected to co-treatment with TNFα/CHX (−) for 6 h or left untreated (−). Western blots using antibodies against Bak (panel 1), PARP (panel 2), and β-actin (panel 3) (relative sample loading). (f, g) Non-silencing siRNA-transfected cells (top panels) and Bak-knockdown cells (bottom panels) were double stained with annexin V-enhanced green fluorescent protein and PI. Fluorescence was detected using a flow cytometer to analyze necrotic in the presence (right column) or absence (left column) of TNFα and CHX for 6 h in A549 (f) and SW480 (g) cells.