Figure 6
Stabilization of tBid and caspase-3 amplification loop contributes to ABT-737 and TRAIL-induced apoptosis. (a) T98G cells were treated for 2 h with 2 ng/ml TRAIL and 5 μM ABT-737 or with 5 ng/ml TRAIL. Then, cells were washed three times to remove all residual TRAIL and incubated with 20 μM zVAD.fmk for indicated times. The tBid protein expression was determined by western blotting. One representative experiment is shown, and similar results were obtained in two independent experiments. (b) U87MG and U118MG cells were transduced with control vector (shCtrl) or a vector containing two different shRNA sequences against Caspase-3 (shC3_1 and shC3_2). Expression of caspase-3 was analyzed by western blotting. 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. Expression of Bid was analyzed by western blot. α-tubulin served as loading control. A representative experiment of three independent experiments is shown. (c) U87MG (left panel) and U118MG (right panel) cells were treated for 24 h with 5 ng/ml (U87MG) or 10 ng/ml (U118MG) TRAIL and/or 5 μM ABT-737. Apoptosis was determined by FACS analysis of DNA fragmentation of propidium iodide-stained nuclei. Data represent mean+S.E.M. of three independent experiments performed in triplicate (*P<0.005 and **P<0.001). (d) U87MG (left panel) and U118MG (right panel) cells were treated for 12 h with 5 ng/ml (U87MG) or 10 ng/ml (U118MG) TRAIL and/or 5 μM ABT-737. Colonies were stained with crystal violet after 18 days and were counted under the microscope. One representative experiment (lower panel) and the percentage of colony numbers compared with untreated control are shown (upper panel). Data represent mean+S.E.M. of three independent experiments (**P<0.001)
