Fig 2: miR-125a induces neuroblastoma (NB) cell apoptosis and differentiation in vitro. NB cell lines were transfected with miR-125a mimic or miR-125a control. (a) miR-125a expression in cells after transfection measured using reverse transcription-quantitative (RT-q) PCR. (b) Cell proliferation examined using cell counting kit-8 (CCK8). (c) Cell apoptosis assessed by Hoechst staining. (d) Immunofluorescence detection of ß-Tubulin-III expression in cells to assess cell differentiation. (e) Protein expression of GAP43, NSE, Bax, and cleaved-caspase-3 in cells examined using western blot. Results are shown as mean ± SD, representative of three technical replicates (*p < .05 vs. miR-125a control by two-way analysis of variance [ANOVA])

Fig 3: The histone deacetylase 2 (HDAC2)/miR-125a/PHOX2B axis influences the apoptosis and differentiation of neuroblastoma (NB) cells in vitro. NB cell lines were cotransfected with miR-125a mimic + HDAC2-OE or miR-125a mimic + PHOX2B-OE. (a) HDAC2 and PHOX2B protein expression in cells was determined using western blot. (b) Cell apoptosis assessed by Hoechst staining. (c) Immunofluorescence detection of ß-Tubulin-III expression in cells to assess cell differentiation. (d) Protein expression of GAP43, NSE, Bax, and cleaved-caspase-3 in cells examined using western blot. Results are shown as mean ± SD, representative of three technical replicates (*p < .05 vs. miR-125a mimic + NC-OE by two-way analysis of variance [ANOVA])

Fig 4: (A): Representative 40× and 100× photomicrographs of lumbar spinal cord ventral grey horn immunostained for GAP-43 protein at week 4 and week 6 post-injury. Note the increase in the intensity of GAP-43 immunoreactivity in the CRUSH compared to all experimental groups. The GAP-43 immunoreactivity number is remarkably less in the CRUSH + SPF-FB(IP) and CRUSH + SPF-FB(S.C.)-treated animals compared to the saline-treated group. (B): Bar graph shows the GAP-43 immunoreactive intensity in the ipsilateral ventral grey horn at week 4 and week 6 post-injury. Note significantly increased staining intensity in the CRUSH, CRUSH + 1X SPF-FB(IP), and CRUSH + 1X SPF-FB(S.C.) groups following nerve injury at Week 4. Whereas, IP and S.C SPF treatments significantly decreased the GAP-43 immunoreactivity at Week 6 post-injury. * indicates p < 0.001 SHAM vs. All groups; ¥ indicates p < 0.002 CRUSH vs. SHAM; epresentative 40× and 100× photomicrographs of lumbar spinal cord indicates p < 0.01 CRUSH + 1X SPF-FB(S.C.) vs. SHAM; ** indicates p < 0.03 CRUSH vs. CRUSH + 1X SPF-FB(IP) and CRUSH + 1X SPF-FB(S.C.). (C): Bar graph shows the intensity of the GAP-43 immunoreactivity in the ipsilateral dorsal grey horn at week 4 and week 6 post-injury. Note significantly increased staining intensity in the CRUSH, CRUSH + 1X SPF-FB(IP) and CRUSH + 1X SPF-FB(S.C.) groups following nerve injury at Week 4. Whereas, IP SPF-FB treatment significantly decreased the GAP-43 immunoreactivity at Week 6 post-injury. * indicates p < 0.0001 SHAM vs. All groups; ¥ indicates p < 0.02 CRUSH vs. CRUSH + 0.5X SPF-FB(IP) and CRUSH + 1X SPF-FB(S.C.). (D): Western blot analysis of GAP-43 in the spinal cord at Week 4 and Week 6 following nerve injury. (E): Bar graph shows significantly increased GAP-43 content in the CRUSH, CRUSH + SPF-FB-treated groups compared to NAÏVE and SHAM groups at Week 4 and Week 6 post-injury. However, IP and S.C. SPF-FB treatments significantly decreased the GAP-43 protein content in the spinal cord compared to CRUSH animals. * indicates p < 0.0001 CRUSH vs. NAÏVE and SHAM; ¥ indicates p < 0.001 CRUSH vs. 0.5 CRUSH + 0.5X SPF-FB(IP); # indicates p < 0.02 CRUSH vs. CRUSH + 1X SPF-FB (S.C.); epresentative 40× and 100× photomicrographs of lumbar spinal cord indicates p < 0.009 CRUSH vs. CRUSH + 2X SPF-FB(S.C.); a indicates p < 0.0001 NAÏVE and SHAM vs. All groups; ** indicates p < 0.048 CRUSH vs. CRUSH + 0.5X SPF-FB(IP).

Fig 5: Effects of NEP1-40 on the expression of GAP-43 and MAP-2 as determined via immunohistochemistry. Representative images of (A and B) GAP-43 and (C and D) MAP-2 expression in the ipsilateral striatum of the sham, MCAO + saline and MCAO + NEP1-40 groups at days 1, 3, 7, 14 and 28 following MCAO (scale bar, 50 µm). Quantitative analysis of (E) GAP-43 and (F) MAP-2 expression in the ipsilateral striatum. *P<0.05 and **P<0.01 vs. sham or day 1; #P<0.05 vs. MCAO + saline. MCAO, middle cerebral artery occlusion; NEP1-40, Nogo extracellular peptide 1-40; GAP-43, growth-associated binding protein 43; MAP-2, microtubule associated protein 2.