Necroptosis is not activated by nor required for Wallerian degeneration. (A) Schematic diagram of axotomy-induced Wallerian degeneration in cultured DRG neurons. (B) To assess whether axotomy activates the necroptotic pathway leading to the phosphorylation of MLKL, axon lysates are collected at 0 and 3 h after axotomy from WT DRG neurons and at 3 h after axotomy from SARM1 KO DRG neurons. 3 h after axotomy was chosen because this is the time point at which SARM1 is activated and severed axons are committed to degenerate. MLKL is not phosphorylated in response to axon transection. (C) To determine if necrostatin-1s blocks Wallerian degeneration, WT DRG neurons were treated with 20 µM necrostatin-1s (Nec-1s) 30 min before axon severing. Axon degeneration was quantified at 0, 9, and 24 h after axotomy. Axons with a DI >0.3 are defined as degenerated (dashed line). Note that necrostatin-1s does not block degeneration of severed axons. Data represent the mean ± SEM, n = 3; two-way ANOVA with post hoc Bonferroni’s multiple comparison test; time, F(1.008, 4.031) = 168.9, P = 0.0002; interaction, F(4, 8) = 2.284, P = 0.1488; *, P < 0.05; **, P < 0.01; and ***, P < 0.001. (D) To determine if genetic loss of MLKL blocks Wallerian degeneration, WT DRG neurons were treated with a control or MLKL targeted shRNA as in S4 and S5. Axon degeneration was quantified at 0, 9, and 24 h after axotomy. Axons with a DI >0.3 are defined as degenerated (dashed line). Knockdown of MLKL does not block degeneration of severed axons. Data represent the mean ± SEM, n = 4; two-way ANOVA with post hoc Bonferroni’s multiple comparison test; time, F(1.142, 6.854) = 230.8, P < 0.0001; interaction, F(6, 12) = 5.801, P = 0.0048; *, P < 0.05; **, P < 0.01; and ***, P < 0.001.