Apoptosis and not necroptosis is triggered by combined loss of cIAPs and XIAP and is dependent on RIPK1, which is critical for tnf transcription, and RIPK3, which is required for TNF secretion. (A) BMDMs of the genotypes indicated were treated for 24 hours with any of the following compounds alone or in combination: the RIPK1 inhibitor Nec-1 (50 μM), the broad-spectrum caspase inhibitor QVD-OPH (10 µM), and the Smac mimetic Comp. A (500 nM). The percentages of dead cells were determined by staining with PI followed by flow cytometric analysis. FLDMs from Ripk1^+/+ and Ripk1^−/− embyros were similarly treated with the exception of Nec-1 (5 µM). LDH release was measured in supernatants 24 hours after treatment. (B) Cleaved caspase-3 detected in lung histology of c1^LCc2^−/−X^−/− mice but not littermate controls (c1^wt/flc2^+/−X^−/−, c1^fl/flc2^−/−X^−/−). The bars represent 50 µm; ×20 magnification, NA 0.8. (C) WT and Ripk3^−/− BMDMs treated with Smac mimetic (Comp. A, 500 nM) for 0-6 hours and lysates assayed for caspase-3 activity. (D) BMDMs of the genotypes indicated were treated with Comp. A (500 nM), anti-TNF (0.1 mg/ml), Nec-1 (5 or 50 µM), and/or QVD-OPH (10 µM) for 24 hours. The levels of TNF in culture supernatants were measured by ELISA. (E) BMDMs of the genotypes indicated were treated for the times indicated with 500 nM Comp. A, either alone or together with 50 µM Nec-1 (as indicated). The levels of TNF mRNA were determined by relative quantitative PCR and expressed relative to WT BMDMs without treatment with Comp. A treatment. (F) WT, Ripk3^−/−, and Tnf^−/− BMDMs treated with Smac mimetic (Comp. A, 500 nM) for 0-7.5 hours. TNF in cell lysate (left) and supernatant (right) was assessed by ELISA, and the latter normalized to amount of protein. In panels A-F, N ≥ 3 experiments with BMDMs or FLDMs from individual mice or embyros of the genotypes indicated. Mean ± SEM are shown. ***P < .001; **P < .01; *P < .05.