XIAP plays an important role in mediating TNFα-dependent apoptosis induced by Smac mimetics. A, MDA-MB-231 cell line was transfected with XIAP siRNA for 48 h, followed by the treatment of SM-122 or SM-164 in combination with TNFα (10 ng/mL) for 48 h. Cell viability was determined by trypan blue dye exclusion. B, HCT 116 XIAP+/+ and XIAP−/− cell lines were treated with SM-122 or SM-164 alone or in combination with TNFα (0.1 ng/mL) as indicated. PARP cleavage and activation of caspase-3 were examined by Western blotting and cell growth inhibition by a WST assay. C, Jurkat cell lines were treated with SM-122, SM-164, TNFα (300 ng/mL) alone, or the combination of SM-122 or SM-164 with TNFα for 24 h. Apoptosis was analyzed by propidium iodide staining/Annexin V double staining using flow cytometry.