Necrostatin-1 inhibits lysis in a RIPK-1-independent manner. (A) HT-29 cells were pretreated without or with 1 μM or 5 μM Nec-1s or 50 μM Nec-1, 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 1-way ANOVA and Tukey posttest (**P < .01; ***P < .001 vs CZT). (B) PMN were pretreated without or with 20 μM Nec-1s or 200 μM Nec-1, and then were either left in buffer alone or challenged with SA, as indicated. After 3 h, LDH in supernatants was measured. Bars represent the average of at least 3 separate experiments ± SEM. P values were determined using a 1-way ANOVA and Tukey posttest (**P < .01; ***P < .0001 vs SA). (C) HT-29 cells were pretreated without or with Nec-1, varied concentrations of GSK’963 (active inhibitor) or of GSK’962 (inactive enantiomer), 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 at least 3 experiments ± SEM. P values were determined using a 1-way ANOVA and Tukey posttest (*P < .05 vs untreated; ^#P < .05 vs GSK’962; ^{^}P < .05 vs CZT). (D) PMN were pretreated without or with Nec-1 or varied concentrations of GSK’963. PMN were either left in buffer alone or challenged with SA as indicated. After 3 h, LDH in supernatants was measured. Bars represent the average of at least 3 separate experiments ± SEM. P values were determined using a 1-way ANOVA and Tukey posttest (*P < .05 vs untreated; ^#P < .05 vs SA). (E) Nonreduced lysates from untreated and CZT-treated HT-29, PMN, and PMN-SA were analyzed by immunoblot for RIPK-1, phosphorylated-RIPK-1, and, in the PMN samples, β-actin. Shown is a representative of 3 separate experiments. Samples were separated in lanes on the same gel, but were noncontiguous. Splicing is denoted with a thin vertical line.