Fig. 3

JL5 downregulation of XIAP enhances TRAIL induced caspase-3 dependent cell death in H1299 cells and JL5 increases cytosolic Smac/Diablo. a-b Immunoblot demonstrated that JL5 decreases expression of XIAP in H1299 cells at 24–48 h but has no effect on (b) A549 cells at 24 h. a,c TAK1 activity directly correlates with the expression of XIAP. c H1299 cells were transfected with mXIAP or control vector, then treated with TRAIL for 3 h. Immunoblot demonstrates mXIAP inhibited TRAIL processing of caspase-3 to its 17 kDa form. d H1299 cells were transfected with siRNA targeting XIAP for 48 h then treated with TRAIL for 3 h. Immunoblot demonstrates that knockdown of XIAP enhanced TRAIL induced processing of caspase-3 to its 19 kDa fragment. e H1299 cells were transfected with mXIAP or control vector for 24 h then treated with JL5 together with TRAIL for 24 h and cell counts performed. Data represents the mean of 4 independent experiments showing mXIAP inhibited cell death induced by JL5 in combination with TRAIL. f Western blot of cytosol demonstrating that JL5 increases cytosolic Smac/DIABLO as early as 3 h in the H1299 cells but not the A549 cells. g-h Immunoblot of cytosol of cells treated with JL5 and TRAIL alone and in combination for 24 h. g Both JL5 and TRAIL increase cytosolic cytochrome c, while (h) JL5 alone increased cytosolic Smac/Diablo in the H1299 cells but not in the A549 cells