DLK inhibition and cytoNMNAT1 expression block axon degeneration induced by dimerization of MLKL.ND. (A) Representative bright-field and TMRM images of axons from WT DRG neurons expressing MLKL.ND at 24 h after BB treatment. Neurons were pretreated with DMSO or 500 nM DLK inhibitor (DLKi; GNE-3511). Scale bar, 50 µm. (B) Quantification of axon degeneration at the indicated times after BB (100 nM) application for the conditions described in A. DLK inhibition blocks axon degeneration for at least 48 h after BB application. Axons with a DI >0.3 are defined as degenerated (dashed line). Data represent the mean ± SEM, n = 4; two-way ANOVA with post hoc Bonferroni’s multiple comparison test; time, F(1.910, 9.552) = 62.13, P < 0.0001; *, P < 0.05; **, P < 0.01; and ***, P < 0.001. (C) Representative bright-field and EthD fluorescent images of neuronal cell bodies for the conditions described in A at 24 h after BB application. Scale bar, 30 µm. (D) Quantification of neuronal cell death (EthD⁺) 24 h after BB treatment for the conditions described in A. DLK inhibitor does not block cell death. Data represent the mean ± SEM, n = 3; two-tailed unpaired t test, P = 0.78. (E) Quantification of axon degeneration in WT neurons expressing MLKL.ND and infected with lentivirus expressing EGFP (FUGW control) the axon survival factor cytoNMNAT1. Expression of cytoNMNAT1 blocks axon degeneration induced by MLKL.ND dimerization. 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(1.863, 14.90) = 144.8, P < 0.0001; interaction, F(8, 16) = 4.061, P = 0.0082; *, P < 0.05; **, P < 0.01; and ***, P < 0.001.