Necrostatin-1s does not prevent axon degeneration following dimerization of MLKL.ND. (A) Representative bright-field and TMRM images for DRG neurons expressing MLKL.ND 24 h after BB addition. The neurons were treated with either DMSO (control) or necrostatin-1s (Nec-1s) 30 min before BB addition. Scale bar, 50 µm. (B) Quantitative analysis of axon degeneration at 0 h and 24 h after BB application from bright-field images for neurons treated with DMSO or the indicated dose of necrostatin-1s. Necrostatin-1s does not block axon degeneration, consistent with MLKL.ND functioning after RIPK1 in the necroptosis pathway. Axons with a DI >0.3 are defined as degenerated (dashed line). Data represent the mean ± SEM, n = 5; two-way ANOVA with post hoc Bonferroni’s multiple comparison test; time, F(1, 9) = 257.4, P < 0.0001; interaction, F(9, 9) = 2.513, P = 0.0930; *, P < 0.05; **, P < 0.01; and ***, P < 0.001.