Fig 2: SCs in the co-culture system. (A) Cell viability assay indicated that the cell viability of SCs co-cultured with ADSCs for 3, 4, 5, 6 and 7 days was significantly higher than those cultured alone. (B) Secretion of neurotrophic factors in the co-culture system. NGF, GDNF, FN and LN in the supernatants of SCs in the co-culture system were significantly higher than in SCs cultured alone, as ELISA revealed. (C) Reverse transcription-polymerase chain reaction demonstrated that mRNA expression levels of neurotrophic factors (NGF, GDNF) and extracellular matrix components (FN, LN) in SCs co-cultured with ADSCs for 14 days were significantly higher than those in SCs cultured alone. Data are expressed as the mean ± standard deviation. *P<0.01 vs. control group. FN, fibronectin; LN, laminin; NGF, nerve growth factor; GDNF, glial cell line-derived neurotrophic factor; SC, Schwann cell; ADSCs, adipose derived stem cells; OD, optical density.

Fig 3: NGF, BDNF and CGRP levels in the bladder tissue. The levels of (A) NGF, (B) BDNF and (C) CGRP in rat bladders were detected by ELISA. *P<0.05 vs. control; #P<0.05 vs. caffeine group; @P<0.05 vs. DM group. NGF, nerve growth factor; BDNF, brain-derived neurotrophic factor; CGRP, calcitonin gene-related peptide; DM, diabetes mellitus.

Fig 4: TGF-ß1 promoted the mRNA expression of NGF in SCDC2 cells in Smad2/3-dependent and p38 MAPK-dependent manners. Effects of (A) SIS3 (10 µM), and (B) SB203580 (10 µM) on expression of NGF mRNA were evaluated as described in Materials and methods. Data represent the mean ± standard deviation (n=6). *P<0.05. (C) Phosphorylation status of Smad2/3 and p38 MAPK in cells stimulated with TGF-ß1 (10 ng/ml) for the indicated times, evaluated using western blot analysis. (D) After 24-h starvation, cells were pretreated with Smad3 inhibitor SIS3 (10 µM) for 30 min and then treated with or without TGF-ß1 (10 ng/ml) for 30 min, and the status of nuclear translocation of Smad2/3 following TGF-ß1 stimulation was examined using immunofluorescence analysis (×200 magnification; scale bar, 50 µm). (E) Phosphorylation status of MAPKAPK-2 evaluated using western blot analysis in cells stimulated with TGF-ß1 (10 ng/ml) and/or with the inhibitor SB203580. (F) Effect of SP600125 (10 µM) on expression of NGF mRNA was evaluated as described in Materials and methods. Data represent the mean ± standard deviation (n=6). *P<0.05. TGF, transforming growth factor; NGF, nerve growth factor; SCDC, single cell-derived culture; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; MAPK, mitogen-activated protein kinase; MAPKAPK-2, MAPK-activated protein kinase 2.

Fig 5: TGF-ß1 promoted the mRNA expression of NGF in SCDC2 cells through its type I receptor in a dose-dependent manner. After 24-h culture in growth medium, SCDC2 cells were starved for 24 h. The starved cells were then treated with (A) TGF-ß1 at various concentrations for 24 h, or (B) pretreated with or without TGF-ß type I receptor inhibitor SB-431542 (10 µM) for 30 min and then with or without TGF-ß1 (10 ng/ml) for 24 h. (C) Starved cells were treated with or without TGF-ß1 (10 ng/ml) for the indicated times. The relative expression level of NGF was evaluated using reverse transcription-quantitative polymerase chain reaction. Data represent the mean ± standard deviation (n=6). *P<0.05. TGF, transforming growth factor; NGF, nerve growth factor; SCDC, single cell-derived culture.