Figure 5
Bid is essential for ABT-737 and TRAIL-induced apoptosis. U87MG and U118MG cells were transduced with control vector (shCtrl) or a vector containing two different shRNA sequences against Bid (shBid_1 and shBid_2). (a) Expression of Bid was analyzed by western blotting. (b) 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. Bax conformational change was determined by IP, Bax expression in lysates served as 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 24 h with 5 ng/ml (U87MG) or 10 ng/ml (U118MG) TRAIL and/or 5 μM ABT-737. Cell viability was determined by MTT assay and is expressed as percentage of untreated controls. Data represent mean+S.E.M. of three independent experiments performed in triplicate. (e) 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)
