Figure 3

TNF and TNFR1 are required for zVAD-fmk/TNC-sc(mu)TNF80 induced cell death. (a) MPCs and macrophages derived thereof were analyzed by flow cytometry with for cell surface expression of the indicated proteins. (b,c) MPCs and macrophages derived from wild type, TNF- (b) and TNFR1-deficient mice (c) were stimulated with the indicated combinations of human TNF (50 ng/ml), TNC-sc(mu)TNF80 (200 ng/ml) and zVAD-fmk (20 μM). After 36 hours cell viability was quantified using the MTT assay or crystal violet staining. Data points derived from 9 (b) or 7 (c) independent experiments together with with mean±S.E.M. are depicted. (d) Macrophages derived from HoxB8-immortalized MPCs of wild type and TNFR2-deficient mice were stimulated overnight with the indicated combinations of TNC-sc(mu)TNF80 (200 ng/ml) and zVAD-fmk (20 μM). Tnf mRNA induction was analyzed by qPCR. Data points of four independent experiments together with mean±S.E.M. are shown. (e) Wild type and TNFR2-deficient macrophages were treated as indicated for 7 h with TNC-sc(mu)TNF80 (200 ng/ml), zVAD-fmk (20 μM) and necrostatin-1 (45 μM). Cells were analyzed by western blotting for the presence of TRAF2 and cIAP1. ***p<0.001