Fig 2: 4-AP increases the number of Schwann cells and the expression of markers of an early SC differentiation state. (a) Representative image of co-immunostaining of S100 (green) and p75-NTR (red) in wound sections and dashed line denotes epidermal/dermal border. Scale bars = 50 μm. (b,c) Quantification of S100+ and p75-NTR+ expressing cells in healed wounds. Data represents 20 images from 5 different mouse wounds and are shown as mean ± SEM, n = 5 animals per group, statistical significance indicated by asterisks (O = saline mouse data points, ∆ = 4-AP treated mouse data points, ** = p between 0.01 and 0.001, and *** = p between 0.001 and 0.0002 vs. saline). (d) Representative image of Western blot of p75-NTR and GAPDH. (e,f) Normalized integrated densities and the ratio of p75-NTR protein expression represented as mean ± SEM, n = 5 animals per group, statistical significance indicated by asterisks (O = saline mouse data points, ∆ = 4-AP treated mouse data points, *** = p between 0.001 and 0.0002 vs. saline).

Fig 3: Verification of enteric glial cell-specific Mpz-EPOR-KO in mice. A Characterization of mouse enteric glial cells (EGCs). The identity and purity of SCs were confirmed using IF staining of GFAP, p75NTR, and S100 under a fluorescent microscope (ZEISS Apotome 2). The purity of the cultured EGCs (99%) was analyzed by double positive staining of DAPI with GFAP/p75NTR/S100 markers from 3 independent experiments. Each image represents 3 images from 3 independent experiments. Scale bar: 100 μm, n = 3. B PCR genotyping with DNA isolated from EGCs (passage zero, EGC-P0; passage one, EGC-P1) revealed no deletion of EPOR in MpzCre-EPORflox/flox mice. EPOR bands of wild-type (control; 390 bp) and flox/flox (428 bp) tail samples of mice are depicted (n = 3). C PCR genotyping with DNA isolated from the segments of the intestine (D, duodenum; J, jejunum; I, ileum) revealed no deletion of EPOR in MpzCre-EPORflox/flox mice (n = 3)

Fig 4: Verification of vagus nerve Schwann cell-specific Mpz-EPOR-KO in mice. A Characterization of mouse vagus nerve derived Schwann cells (VNSCs). The identity and purity of SCs were confirmed using IF staining of S100, p75NTR, and Mpz under a fluorescent microscope (ZEISS Apotome 2). The purity of the cultured SCs (99%) was analyzed by double positive staining of DAPI with S100/p75NTR/Mpz markers from 3 independent experiments. Each image represents 3 images from 3 independent experiments. Scale bar: 50 μm, n = 3. B PCR genotyping with DNA isolated from the vagus nerve and VNSCs (passage zero, SC-P0; passage one, SC-P1) revealed the presence of the 220-bp PCR product, resulting from MpzCre mediated recombination in the Schwann cell of MpzCre-EPORflox/flox mice and not in wild-type (control; 390 bp) and flox/flox (428 bp) mice (n = 3)