Fig 1: Neurotrophic secretion of rat ADSCs. (A) Immunocytochemistry indicated neurotrophic factors as NGF (panel a), GDNF (panel b), fibronectin (panel c) and laminin (panel d) could be detected in rat ADSCs and supernatants of cultured medium. Green fluorescence indicates the protein of interest, and blue fluorescence indicates nuclei. (B) ELISA results were consistent. Data are expressed as the mean ± standard deviation. *P<0.01 vs. control group. ADSCs, adipose derived stem cells.
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.
Supplier Page from Abcam for Rat beta NGF ELISA Kit