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. 2018 Feb 23;9:306. doi: 10.3389/fimmu.2018.00306

Figure 5.

Figure 5

Mixed lineage kinase domain-like protein (MLKL) cooperates with RIPK3 during complement-dependent cytotoxicity (CDC) but not with receptor-interacting protein kinase 1 (RIPK1). (A) K562 cells were pretreated with GSK’872 and/or necrosulfonamide (NSA) in the indicated concentrations or with DMSO as control (0), for 1 h at 37°C, and then treated with antibody and complement for 1 h min at 37°C. The percentage of cell death (CD) was measured by propidium iodide (PI) inclusion. Results are expressed as the mean percentage of the inhibition of CD ± SD and represent three independent experiments. *P < 0.01 relative to pretreatment with 0.25 µM GSK’872 without NSA or to 2 µM NSA without GSK’872. [% Inhibition = (% Death of Control − % Death of Treatment)/%Death of Control × 100]. (B) K562 cells were transfected with Smart Pool siRNA targeting MLKL (siRNA) or scrambled siRNA (Sc) as a control by electroporation. After 24 h, the cells were treated with GSK’872 at the indicated concentrations (or DMSO as control, 0) and then with antibody and complement for 1 h at 37°C. CD was measured as above. Statistical analysis showed that CDC of MLKL knocked down cells is significantly less sensitive to GSK’872 than is CDC of cells treated with a scrambled siRNA (two-way-ANOVA, P < 0.001). (C) WT and MLKL KO mouse embryonic fibroblasts (MEFs) were treated with necrostatin-1 (Nec-1s), a selective RIPK1 inhibitor, in increasing concentrations, or with DMSO as control, for 1 h followed by incubation with 10% normal human serum (NHS). The lysis percentage was determined by PI inclusion. Results are presented as the mean percentage of lysis ± SD. Statistical analysis showed that MLKL KO MEF is as sensitive to CDC as WT MEF (two-way-ANOVA, P = 0.538). (D) MLKL-silenced K562 cells were treated with Nec-1s at the indicated concentrations and then with antibody diluted 1:18 or 1:15 for scrambled or siRNA-treated cells, respectively, followed by complement (NHS, 50%). CD was measured by PI staining, expressed as a calculated mean percentage ± SD, representing three independent experiments. Statistical analysis showed that CDC of MLKL knocked down cells is as sensitive to Nec-1 as CDC of cells treated with a scrambled siRNA (two-way-ANOVA, P = 0.104).