A20 inhibits caspase-8 cleavage and TRAIL-induced apoptosis. A20 inhibits caspase-8 cleavage and TRAIL-induced apoptosis. A, TRAIL-resistant LN443 cells were transfected with A20, TRAF2, and scrambled siRNA; treated with TRAIL for 24 hours; and examined for cell death (points: means; bars: SE; n = 6; ***P < 0.001). B, the transfected cells were treated with 100 ng/mL TRAIL and examined by caspase-8 (Casp-8) enzymatic activity assay (points: means; bars: SE; n = 6; ***P < 0.001; NS, no significance). C, apoptotic cell death was observed under phase contrast microscopy in A20 siRNA but not scrambled and TRAF2 siRNA-transfected cells. D, LN443 cells were transfected with siRNA, treated with 100 ng/mL TRAIL, and examined by immunoblotting for the knockdown of the proteins and the cleavage of caspase-8 and RIP1. Nontransfected TRAIL-sensitive U343MG cells were included as the control for caspase-8 and RIP1 cleavage. E, the DISC was isolated from the LN443 cells transfected with siRNA and, together with the cell lysates, examined by immunoblotting for caspase-8 cleavage. F, LN443 cells transfected with A20 and RIP1 siRNA were treated or untreated with 100 ng/mL TRAIL in the absence or presence of the caspase-8 inhibitor z-IEDT and examined by immunoblotting for caspase-8 cleavage. Anita C. Bellail et al. Cancer Discovery 2012;2:140-155 ©2012 by American Association for Cancer Research