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. 2018 Dec 10;115(52):E12417–E12426. doi: 10.1073/pnas.1812518115

Fig. 1.

Fig. 1.

DNA methylation is required for axon regeneration. (A) Representative longitudinal sections of sciatic nerve from DMSO or RG108 treated mice. Nerves were collected 3 d after SNI and sections were stained for SCG10. Dotted lines indicate the crush site. Arrowheads point to the longest axons. (B) In vivo axon regeneration was calculated from images in A. The number of regenerating axons stained with SCG10 was measured at various distances from the crush site (n = 5 biological replicates for each, respectively; **P < 0.01 by one-way ANOVA; mean ± SEM). (C) Adult DRG neurons dissected from DMSO- or RG108-treated mice that received or not a prior (3 d) SNI were cultured for 20 h and stained with the neuronal-specific marker TUJ1. (D) Average axon length per soma was calculated from images in C. Total neurite length was measured and normalized to the number of neuronal cell soma to calculate the average axon length (n = 4 biological replicates for each; *P < 0.05, ns, not significant by one-way ANOVA; mean ± SEM). (E) Measurement of global DNA methylation. The global DNA methylation assay was performed using the MethylFlash Methylated DNA 5-mC Quantification Kit with genomic DNA from whole DRG 3 d after SNI (n = 4–5 biological replicates for each; *P < 0.05, **P < 0.01 by one-way ANOVA; mean ± SEM). (F) DRG spot-cultured neurons were treated with DMSO or RG108 (100 μM) 10 min before injury and 24 h later. DRG spot-cultured neurons were fixed and stained for SCG10 40 h after axotomy. Arrow and dotted lines indicate the axotomy site. (G) Regenerating axon length of DRG spot-cultured neurons was measured 40 h after axotomy from images in F (Individual data plotted; n = 8 biological replicates; ****P < 0.0001 by t test; mean ± SEM). (H) DRG spot-cultured neurons were treated at DIV7 and DIV8 with DMSO or 100 μM RG108. Axon lengths of DRG spot-cultured neurons were measured 40 h after initiation of the treatment (n = 6–8 biological replicates for each, ns, not significant by t test; mean ± SEM). (Scale bars A, 500 μm; C, 100 μm; F, 200 μm.)