Figure 4

ABT-737 and TRAIL cooperate to promote mitochondrial accumulation of tBid and mitochondrial outer membrane permeabilization. (a–c) U87MG (left panel) and U118MG (right panel) cells were treated for 4 h with 5 ng/ml (U87MG) or 10 ng/ml (U118MG) TRAIL and/or 5 μM ABT-737. Bid expression was analyzed in whole-cell lysates by western blotting (a) or in cytosolic and mitochondrial fractions as described in Materials and Methods (b). In (c), Bax conformational change was determined by IP; Bax expression in lysates served as control. (d) U87MG (left panel) and U118MG (right panel) cells were treated for indicated times with 5 ng/ml (U87MG) or 10 ng/ml (U118MG) TRAIL and/or 5 μM ABT-737. Mitochondrial transmembrane potential was assessed by FACS analysis. The percentage of cells with loss of mitochondria potential with mean+S.E.M. of three independent experiments in triplicate are shown (*P<0.005 and **P<0.001). (e) U87MG (left panel) and U118MG (right panel) cells were treated for 6 h with 5 ng/ml (U87MG) or 10 ng/ml (U118MG) TRAIL and/or 5 μM ABT-737. Expression of cytochrome-c and Smac in cytosolic and mitochondrial fractions was determined by western blot analysis. CoxIV and α-tubulin served as purity and loading controls of the fractions. In (a–c and e), representative experiments of three independent experiments are shown