Fig 2: SnoN Promotes Neurite Outgrowth From Adult DRG Cultures.(A) Western blot from HEK293 cells electroporated to express GFP, wild-type SnoN, SnoN-DBM, GFP fused SnoN, and GFP fused SnoN-DBM. Cells transfected with wild-type SnoN and SnoN-DBM demonstrate SnoN immunoreactive bands at ~75 kD (lane 3 and 4). Cells transfected with GFP-SnoN and GFP-SnoN-DBM demonstrate SnoN immunoreactive bands and corresponding GFP immunoreactive bands at ~110 kD, a molecular weight consistent with expression a GFP fused variant. (B) Neurite outgrowth from control adult DRG neurons in vitro. NF200 label. (C) Overexpression of SnoN increases neurite length, quantified in E. Infected cells express GFP and are labeled for NF200 (shown). (D) Overexpression of SnoN-D-box mutation further amplifies neurite growth. (E) Quantification confirms that SnoN expression in adult DRG neurons significantly enhances neurite growth (ANOVA p<0.001; post hoc Fisher’s ***p<0.001 comparing treated groups to GFP, and **p<0.01 comparing SnoN to SnoN-DBM group).

Fig 3: Quantification of SnoN-Induced Axonal Regeneration.Quantification reveals a 3-fold increase in the number of axons regenerating into the lesion site among animals treated with SnoN-DBM. * p<0.001.

Fig 4: SnoN Overexpression Overcomes TGF-ß1 Inhibition.(A) Neurite outgrowth in primary adult DRG neurons in the presence of TGF-ß1. Cells expressing GFP and NF200 (shown) are quantified. (B) Overexpression of SnoN in neurons appears to increase neurite length even in presence of TGF-ß1, quantified in D. (C) Overexpression of SnoN-D-box mutation also appears to increase neurite growth. (D) Quantification reveals that overexpression of TGF-ß1 overcomes TGFß-1-mediated inhibition (ANOVA p<0.001; post hoc Fisher’s *p<0.05, **p<0.01).

Fig 5: SnoN Promotes Axonal Regeneration After Spinal Cord Injury.Sensory axonal regeneration into site of C3 dorsal column lesion. (A, C) Animal treated with SnoN-DBM exhibits regeneration into lesion site. Boxed region is magnified in C, a region entirely with the lesion site. Axons exhibit circuitous trajectories and make abrupt turns, consistent with regenerating axons (Tuszynski, 2012). Dashed lines indicate the caudal lesion/host interface. (B, D) Control subjects exhibit few axons within lesion site. Boxed region is entirely within the lesion site and is shown in D. (E) Another animal that received SnoN-DBM. Several axons are present in the central-distal aspect of lesion site, shown at higher magnification in F (arrow). Lesion margins are indicated by dashed lines. Significantly greater numbers of axons penetrate SnoN-DBM treated animals (P < 0.005; see text). Scale bar A, B: 250 µm; C, D: 100 µm; E, 175 µm; F, 50 µm.