Figure 3

Mlkl is required for necroptosis in MEFs and macrophages. (A) Peritoneal macrophages from wild-type and Mlkl^−/− mice were treated as indicated for 24 h with DMSO, LPS (100 ng/ml), zVAD (20 μM), oxLDL (150 μg/ml), LPS+zVAD, or oxLDL+zVAD. Cell viability was determined by Annexin V-Propidium Iodide double-staining. Necrostatin-1 (Nec-1, 30 μM) was included in some samples as a positive control of necroptosis inhibition. Data are expressed as mean ± SEM of triplicates. ^***P < 0.001 by Student's t-test. (B) Wild-type and Mlkl^−/− MEFs were treated with DMSO, TNF (30 ng/ml), CHX (10 μg/ml), zVAD (20 μM), TNF+CHX, or TNF+CHX+zVAD for 8 h. Cell viability was determined by MTT assay. Data are expressed as mean ± SEM of triplicates. ^***P < 0.001 by Student's t-test. (C) Wild-type and Mlkl^−/− MEFs were treated as indicated for 24 h with DMSO, TNF (30 ng/ml), Smac mimetic (SmacM) (10 nM), zVAD (20 μM), TNF+SmacM, or TNF+SmacM+zVAD. Cell viability was determined by MTT assay. Data are expressed as mean ± SEM of triplicates. ^***P < 0.001 by Student's t-test. (D) Wild-type and Mlkl^−/− MEFs were treated with DMSO, Etoposide (50 μM), staurosporine (STS) (500 nM), TRAIL (20 ng/ml), CHX (10 μg/ml), zVAD (20 μM), or TRAIL+CHX, TRAIL+CHX+zVAD as indicated for 24 h. Cell viability was determined by MTT assay. Data are expressed as mean ± SEM of triplicates. ^***P < 0.001 by Student's t-test.