Fig. 2. TNFR2-induced cell death in xiap^−/− macrophages requires soluble TNF and TNFR1 activation.

a BMDMs from WT and xiap^−/− were treated with TNC-TNF and/or anti-TNFα (100 ng/ml) after 4 h of stimulation, and after 24 h cell death was measured via PI uptake by flow cytometry. b, c BMDMs from WT, xiap^−/−, xiap^−/− tnf^−/−, xiap^−/− tnfr1^−/− and xiap⁺ nfr2^−/− were treated with either TR1-TNF or TNC-TNF. d Representative western blot shows that TRAF2 degrades in both WT and xiap^−/− BMDMs treated with TNC-TNF. e Representative western blot shows loss of cIAP1 and XIAP in macrophage-specific (LC) genotypes. Blots are representative of three independent experiments. f WT, xiap^−/−, xiap^−/−ciap1^LC and ciap1^LCciap2^−/− BMDMs were treated with TR1-TNF, TNC-TNF, either a combination of TR1- and TNC-TNF or an initial stimulation with TNC-TNF for 4 h and subsequent TR1-TNF stimulation. Data shown are mean ± SEM including n = 3–5 biological replicates. Experiments were repeated at least three times independently. Statistical significance was calculated using two-way ANOVA with **p < 0.01, ***p < 0.001 and ****p < 0.0001.