Figure 2. XIAP limits LPS- and TNF-induced apoptosis and necroptosis in macrophages.

(a,b) WT and x^−/− BMDM were pre-incubated with or without (a) LPS (20 ng ml⁻¹) or (b) TNF (100 ng ml⁻¹) for 2–3 h and were cultured with IAP antagonists of differing IAP specificities (500 nM; see Fig. 1g) as indicated for 24 h. Cell death was assessed by flow cytometric analysis of PI uptake. Data are presented as the % Dead cells, n=3 mice, mean+s.e.m., one of two experiments. (c,d) WT, c1^LysMcrec2^−/− or c1^LysMcrex^−/−c2^−/− BMDM were stimulated with (c) LPS (20 ng ml⁻¹) or (d) TNF (100 ng ml⁻¹) and cell death (% Dead cells) measured by flow cytometric analysis of PI uptake. n=3 mice, mean+s.e.m., one of three experiments. (e) WT, c1^LysMcrec2^−/− or c1^LysMcrex^−/−c2^−/− BMDM were stimulated with LPS (20 ng ml⁻¹) or TNF (100 ng ml⁻¹) and lysates were analyzed for caspase-8 processing by immunoblot as indicated. Representative of one of two experiments. Full-size immunoblots are presented in Supplementary Fig. 10. (f) WT, x^−/− and x^−/−c2^−/− BMDM were primed with LPS (20 ng ml⁻¹) or TNF (100 ng ml⁻¹) and cultured with cIAP1/2-selective antagonist, 711 (500 nM), as indicated, and lysates analyzed for caspase-8 processing by immunoblot. Representative of one of three experiments. Full-size immunoblots are presented in Supplementary Fig. 10. (g) WT, x^−/−, or x^−/−c2^−/− BMDM were primed for 3 h with LPS (20 ng ml⁻¹) or TNF (100 ng ml⁻¹), and as indicated cultured with the cIAP1/2-selective antagonist, 711 (500 nM), in the presence or absence of Q-VD-OPh (20 μM, added in the last 20 min of priming). Cell death was measured after 24 h by PI uptake. n=3 mice, mean+s.e.m., one of two experiments. (h) WT, Mlkl^−/− and Ripk3^−/− BMDM were primed for 3 h with LPS and treated with Q-VD-OPh (20 μM) as indicated for the final 20 min prior to addition of Cp.A (500 nM). Cell death was measured by assaying lactate dehydrogenase (LDH) release (n=3 mice per genotype). (i) Cell lysates of WT, Mlkl^−/− and Ripk3^−/− BMDM primed with LPS for 3 h and treated with Cp.A (500 nM) for 6 h were analyzed by immunoblot. Representative immunoblot analysis of three mice of each genotype. Full-size immunoblots are presented in Supplementary Fig. 10. (j) WT, Ripk3^−/−, Mlkl^−/− and Ripk3^−/−Caspase-8^−/− BMDM were primed with Pam₃Cys (2.5 μg ml⁻¹) for 3 h, treated with Q-VD-OPh in the final 20 min of priming, and Cp.A added, as specified, for 24 h. In some cases RIP3 kinase inhibitor (R3 inhib, GSK872; 1 μM) was added 20 min prior to the addition of Cp.A. Cell death was measured by PI uptake and flow cytometric analysis (% Dead cells). n=3 mice, mean+s.e.m.