Heme-induced cell death requires the RIP proteins. (A) BMM (2 × 105/well) from Tlr4−/− mice were left untreated (−) or stimulated with heme at 30μM, z-VAD-fmk at 10μM and/or TNF at 0.5 ng/mL (+) for 6 hours and supernatants were collected for LDH determination. (B) Peritoneal macrophages (2 × 105/well) from C57Bl/6 mice were treated for 6 hours with heme at 30μM (+) in the presence or absence of necrostatin-1 (Nec-1) at 25 and 50μM. (C) Experiment performed as in (A) with Nec-1 at 50μM. (D) Wt and Rip1−/− NIH-3T3 fibroblasts and (E) peritoneal macrophages from WT and Rip3−/− were left untreated (−) or stimulated with heme at 50 or 100μM in the absence or presence of TNF at 0.5 ng/mL for 6 hours and supernatants were collected for LDH determination. Data are representative of 3 independent experiments performed in triplicates and represent mean ± SEM (*P ≤ .05; **P ≤ .001; ***P ≤ .0001). Heme induced RIP3 phosphorylation. (F) Peritoneal macrophages from C57BL/6 mice were stimulated with heme (30μM) for the indicated times. (G) Cell lysates from samples treated with heme for 60 minutes were treated with CIP or lambda phosphatase (λPP) for 3 hours. Whole cell extracts were submitted to SDS-PAGE and RIP3 phosphorylation and RIP1 were detected by immunoblotting.
