In vitro sensory axon regeneration of adult rat DRG explants and human iPSC-derived sensory neurons after axotomy in the presence of inhibitors. The inhibitors used were MLN492, NEDD8-mediated ubiquitination inhibitor; NSC697923, K63-ubiquitination inhibitor; MG132, proteasome inhibitor; 3MA, Class III PI3K autophagy inhibitor; D4476, casein kinase 1 inhibitor. A, B, For axotomized axons (A) in adult rat DRG explants, all the five inhibitors exhibited various degrees but significant inhibition to axon regeneration, with the greatest effect observed by 3MA and the most modest effect by MG132. For uncut axons (B), all inhibitors, except 3MA, did not affect continuous axon growth. Each point represents one experiment involving at least three DRGs with multiple cut axons. Analysis was performed using one-way ANOVA with post hoc test. A p-value of <0.05 was considered to be statistically significant. *p-value < 0.05, **p-value < 0.01, ***p-value < 0.001. C, Adult rat DRG explant neurites immediately and 2 h after axotomy by a glass-pulled pipette where the demarcation line is shown clearly (black dotted line). Immediately after axotomy, retraction bulbs (magenta asterisks) can be seen for Neurites 1, 2, 3, and 4. Two hours after axotomy, Neurites 1, 3, and 4 regenerated with the formation of growth cones (green arrows), and axon branching (green hash sign) can be seen for Neurite 4. D, The percentage of human iPSC-derived sensory axons regenerating at 6, 12, and 24 h after laser axotomy, and regeneration was inhibited by all the inhibitors except NSC697923. Analysis was performed using Fisher's exact test. The p-values were then analyzed with the “Analyze a stack of p-values” function in GraphPad Prism with a Bonferroni–Dunn pairwise comparison. E, An aggregate of human iPSC-derived sensory neurons with halo of axons, after 21 d of maturation. F, The results of laser axotomy of the halo of axons. After axotomy, the axons retracted (black arrows) followed by various degrees of regeneration in the presence of inhibitors in the post-24 h.