Lysis of PMN-SA is MLKL-independent. (A) HT-29 cells were pretreated with 0, 0.5, or 5 μM NSA and then stimulated with or without CHX, zVAD-fmk, and TNFα (CZT), as described in “Methods.” After 22 hours, LDH in supernatants was measured. Bars represent the mean of 3 experiments ± SEM. P values were determined using a repeated measures 1-way ANOVA and Tukey posttest (*P < .05 vs CZT and ^#P < .05 vs 0.5 μM NSA). (B) PMN were pretreated with or without 0, 0.5, or 5 μM NSA, and then left in buffer alone or challenged with SA, as described in “Methods.” After 3 h, LDH in supernatants was analyzed. Bars represent the average of at least 3 separate experiments ± SEM. (C) Nonreduced lysates from treated and untreated HT-29 cells, PMN, and PMN-SA were immunoblotted and probed for MLKL, phosphorylated-MLKL, and, in the PMN samples, β-actin. Shown is a representative of 3 experiments. Samples were separated in lanes on the same gel, but were noncontiguous. Splicing is denoted with a thin vertical line.