Journal: International Journal of Molecular Medicine

Article Title: IL-1β and TNF-α suppress TGF-β-promoted NGF expression in periodontal ligament-derived fibroblasts through inactivation of TGF-β-induced Smad2/3- and p38 MAPK-mediated signals

doi: 10.3892/ijmm_2018.3714

Figure Lengend Snippet: TGF-β1 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-β1 at various concentrations for 24 h, or (B) pretreated with or without TGF-β type I receptor inhibitor SB-431542 (10 µ M) for 30 min and then with or without TGF-β1 (10 ng/ml) for 24 h. (C) Starved cells were treated with or without TGF-β1 (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.

Article Snippet: Recombinant human TGF-β1 was obtained from PeproTech, Inc. (Rocky Hill, NJ, USA), recombinant rat NGF was obtained from Alomone Labs (Jerusalem, Israel), and recombinant rat IL-1β and TNF-α were purchased from Miltenyi Biotec GmbH (Bergisch Gladbach, Germany).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Standard Deviation, Derivative Assay

Journal: International Journal of Molecular Medicine

Article Title: IL-1β and TNF-α suppress TGF-β-promoted NGF expression in periodontal ligament-derived fibroblasts through inactivation of TGF-β-induced Smad2/3- and p38 MAPK-mediated signals

doi: 10.3892/ijmm_2018.3714

Figure Lengend Snippet: TGF-β1 promoted the mRNA expression of NGF in SCDC2 cells in Smad2/3-dependent and p38 MAPK-dependent manners. Effects of (A) SIS3 (10 µ M), and (B) SB203580 (10 µ M) 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-β1 (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 µ M) for 30 min and then treated with or without TGF-β1 (10 ng/ml) for 30 min, and the status of nuclear translocation of Smad2/3 following TGF-β1 stimulation was examined using immunofluorescence analysis (×200 magnification; scale bar, 50 µ m). (E) Phosphorylation status of MAPKAPK-2 evaluated using western blot analysis in cells stimulated with TGF-β1 (10 ng/ml) and/or with the inhibitor SB203580. (F) Effect of SP600125 (10 µ M) 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.

Article Snippet: Recombinant human TGF-β1 was obtained from PeproTech, Inc. (Rocky Hill, NJ, USA), recombinant rat NGF was obtained from Alomone Labs (Jerusalem, Israel), and recombinant rat IL-1β and TNF-α were purchased from Miltenyi Biotec GmbH (Bergisch Gladbach, Germany).

Techniques: Expressing, Standard Deviation, Western Blot, Translocation Assay, Immunofluorescence, Derivative Assay, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

Journal: International Journal of Molecular Medicine

Article Title: IL-1β and TNF-α suppress TGF-β-promoted NGF expression in periodontal ligament-derived fibroblasts through inactivation of TGF-β-induced Smad2/3- and p38 MAPK-mediated signals

doi: 10.3892/ijmm_2018.3714

Figure Lengend Snippet: IL-1β and TNF-α suppressed the TGF-β1-induced mRNA expression of NGF in SCDC2 cells by abrogating Smad2/3 and p38 MAPK activities. The effects of IL-1β and TNF-α on TGF-β1-induced mRNA expression of NGF in SCDC2 cells were evaluated using RT-qPCR. The cells were treated with or without (A) IL-1β alone or (B) TNF-α alone at indicated concentrations, (C) TGF-β1 (10 ng/ml) and/or IL-1β (10 ng/ml), and (D) TGF-β1 (10 ng/ml) and/or TNF-α (10 ng/ml). Data represent the mean ± standard deviation (n=6). * P<0.05. Phosphorylation status of (E) Smad2/3 and (F) p38 MAPK was evaluated using western blot analysis in cells treated with or without TGF-β1 (10 ng/ml) alone, TGF-β1 (10 ng/ml) + IL-1β (10 ng/ml), or TGF-β1 (10 ng/ml) + TNF-α (10 ng/ml) for the indicated times. (G) NGF protein concentration secreted into the culture medium was determined using ELISA in cells cultured with or without TGF-β1 (10 ng/ml) alone, TGF-β1 (10 ng/ml) + IL-1β (10 ng/ml), or TGF-β1 (10 ng/ml) + TNF-α (10 ng/ml) for 5 days. (H) Nuclear translocation status of NF-κB p65 (red) was evaluated using immunofluorescence analysis (blue, nuclei; green, filamentous actin) in SCDC2 cells treated with or without IL-1β (10 ng/ml) or TNF-α (10 ng/ml) for 24 h (×200 magnification; scale bar, 50 µ m). IL, interleukin; TNF, tumor necrosis factor; TGF, transforming growth factor; NGF, nerve growth factor; SCDC, single cell-derived culture; MAPK, mitogen-activated protein kinase.

Article Snippet: Recombinant human TGF-β1 was obtained from PeproTech, Inc. (Rocky Hill, NJ, USA), recombinant rat NGF was obtained from Alomone Labs (Jerusalem, Israel), and recombinant rat IL-1β and TNF-α were purchased from Miltenyi Biotec GmbH (Bergisch Gladbach, Germany).

Techniques: Expressing, Quantitative RT-PCR, Standard Deviation, Western Blot, Protein Concentration, Enzyme-linked Immunosorbent Assay, Cell Culture, Translocation Assay, Immunofluorescence, Derivative Assay

Journal: International Journal of Molecular Medicine

Article Title: IL-1β and TNF-α suppress TGF-β-promoted NGF expression in periodontal ligament-derived fibroblasts through inactivation of TGF-β-induced Smad2/3- and p38 MAPK-mediated signals

doi: 10.3892/ijmm_2018.3714

Figure Lengend Snippet: Nerve growth factor secreted by SCDC2 cells following TGF-β1 stimulation promoted neurite extension from the surface of ATPγS-treated PC12 cells. (A) Neurite extension of PC12 cells was visualized by immunostaining (×200 magnification; scale bar, 50 µ m) with anti-neurofilament H antibody (red) and nuclei were stained with DAPI (blue). SCDC2 cells (2×10 4 cells) and rat pheochromocytoma cells PC12 (1×10 4 cells) were co-cultured and treated with or without TGF-β1 (10 ng/ml) for 4 days. Cells were also treated with TGF-β type I receptor inhibitor SB-431542 (10 µ M), TrkA inhibitor GW441756 (2 nM), IL-1β (10 ng/ml), or TNF-α (10 ng/ml) from the beginning of the co-culture. In addition, ATPγS (100 µ M) was added to all cultures during cell seeding. Dimethyl sulfoxide was added to cell cultures as a vehicle control for SB-431542 and GW441756, respectively. (B) Statistical assessment of neurite extension in PC12 cells co-cultured with SCDC2 cells. Data represent the mean ± standard deviation (n=8). * P<0.05. TGF, transforming growth factor; SCDC, single cell-derived culture; IL, interleukin; TNF, tumor necrosis factor; ATPγS, adenosine 5′-O-(3-thio)triphosphate; TrkA, tropomyosin receptor kinase A.

Article Snippet: Recombinant human TGF-β1 was obtained from PeproTech, Inc. (Rocky Hill, NJ, USA), recombinant rat NGF was obtained from Alomone Labs (Jerusalem, Israel), and recombinant rat IL-1β and TNF-α were purchased from Miltenyi Biotec GmbH (Bergisch Gladbach, Germany).

Techniques: Immunostaining, Staining, Cell Culture, Co-Culture Assay, Standard Deviation, Derivative Assay

Journal: International Journal of Molecular Medicine

Article Title: IL-1β and TNF-α suppress TGF-β-promoted NGF expression in periodontal ligament-derived fibroblasts through inactivation of TGF-β-induced Smad2/3- and p38 MAPK-mediated signals

doi: 10.3892/ijmm_2018.3714

Figure Lengend Snippet: Nerve growth factor secreted by SCDC2 cells subsequent to TGF-β1 stimulation promoted the expression of TH mRNA in PC12 cells. SCDC2 cells (7×10 4 cells) and rat pheochromocytoma cells PC12 cells (3.5×10 4 cells) were co-cultured and stimulated with or without TGF-β1 (10 ng/ml) for 24 h. The relative expression level of TH was evaluated using reverse transcription-quantitative polymerase chain reaction in cells also treated with (A) ATPγS (100 µ M), and with (B) TGF-β type I receptor inhibitor SB-431542 (10 µ M), TrkA inhibitor GW441756 (2 nM), IL-1β (10 ng/ml) or TNF-α (10 ng/ml) during the co-culture. Dimethyl sulfoxide was added to cell cultures as a vehicle control for SB-431542 and GW441756, respectively. Data represent the mean ± standard deviation (n=6). * P<0.05. TGF, transforming growth factor; SCDC, single cell-derived culture; IL, interleukin; TNF, tumor necrosis factor; ATPγS, adenosine 5′-O-(3-thio)triphosphate; TrkA, tropomyosin receptor kinase A; TH, tyrosine hydroxylase.

Article Snippet: Recombinant human TGF-β1 was obtained from PeproTech, Inc. (Rocky Hill, NJ, USA), recombinant rat NGF was obtained from Alomone Labs (Jerusalem, Israel), and recombinant rat IL-1β and TNF-α were purchased from Miltenyi Biotec GmbH (Bergisch Gladbach, Germany).

Techniques: Expressing, Cell Culture, Real-time Polymerase Chain Reaction, Co-Culture Assay, Standard Deviation, Derivative Assay