Journal: Physiological Reports

Article Title: Influence of bacterial components on the developmental programming of enteric neurons

doi: 10.14814/phy2.14611

Figure Lengend Snippet: Characterization of ENCDC cultures. Flow cytometry of ENCDC cultures stained with antibodies against the immature neuronal marker, p75 NTR+ ‐FITC demonstrated 95.1% p75 NTR+ ‐positive, 97.3% viability in subculture 3, and 96.1% p75 NTR+ ‐positive, 98.2% viability in subculture 4 (a). Proportion of ENCDC cultures characterized for expression of p75NTR+, pH3, 5‐HT, nNOS, and TH. Scale bar = 20 µm (b). There was a significant increase in the proportion of serotonergic neurons and nitrergic neurons in subculture 5 (gray) compared to subculture 4 (black), but no significant difference in proliferating cells or dopaminergic neurons across the cultures. *p ≤ .05. Values are presented as mean ± SEM (c)

Article Snippet: Single cells were incubated with primary antibody rabbit anti‐mouse p75 NTR+ for 1 hr (1:50, Alomone ANT‐007), and secondary antibody anti‐rabbit IgG microbeads for 15 min (150 µl per 10 7 cells; MACS Miltenyi Biotec 130‐048‐602).

Techniques: Flow Cytometry, Staining, Marker, Expressing

Journal: Scientific Reports

Article Title: Validation of antibodies for the specific detection of human TRPA1

doi: 10.1038/s41598-019-55133-7

Figure Lengend Snippet: Anti-hTRPA1 antibodies studied.

Article Snippet: ACC- 037 , , AN1702 AN1202 AN1150 , 1st extracellular loop AA 747-760 , Alomone Labs , Polyclonal rabbit anti hTRPA1 , 4.5 , ICC, IHC, IP, and WB , 11.

Techniques: Enzyme-linked Immunosorbent 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 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

Journal: Acta physiologica (Oxford, England)

Article Title: Orexin A increases sympathetic nerve activity through promoting expression of proinflammatory cytokines in Sprague Dawley rats

doi: 10.1111/apha.12963

Figure Lengend Snippet: Central administration of orexin A stimulates the expression of proinflammatory cytokines (PICs) in the PVN of SD rats. Real-time PCR analysis of paraventricular nucleus (PVN) PICs mRNA expression levels at 3 hours following acute intracerebroventricular (ICV) infusion of vehicle control (0.9% saline) or orexin A (0.2 nmol) using male adult SD rats. ICV injection of orexin A increases PVN IL-1-β, IL-6, TNF-α and Fra1 mRNA expression in SD rats. The mRNA level in the control sample was assigned to be arbitrary unit (a.u). *P < .05 vs vehicle control; n = 6 each group

Article Snippet: Primary antibodies rabbit anti-OX1R and rabbit anti-OX2R were products of Alomone laboratories (Jerusalem, Israel); mouse anti-Fra1 antibody and mouse anti-TNFα antibody were from Santa Cruz Biotechnology.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Injection

Journal: Acta physiologica (Oxford, England)

Article Title: Orexin A increases sympathetic nerve activity through promoting expression of proinflammatory cytokines in Sprague Dawley rats

doi: 10.1111/apha.12963

Figure Lengend Snippet: Colocalization of OX1R and Fra1 in the paraventricular nucleus (PVN). (A) a representative micrograph showing immunoreactivity of OX1R (green), Fra1 (red) and merged image in the PVN of vehicle control rat; (B) a representative micrograph showing immunoreactivity of OX1R (green), Fra1(red) and merged image in the PVN of central administration of orexin A SD rat. The brain coronal sections were taken from bregma −1.6 mm. 3V, the third ventricle

Article Snippet: Primary antibodies rabbit anti-OX1R and rabbit anti-OX2R were products of Alomone laboratories (Jerusalem, Israel); mouse anti-Fra1 antibody and mouse anti-TNFα antibody were from Santa Cruz Biotechnology.

Techniques:

Journal: Acta physiologica (Oxford, England)

Article Title: Orexin A increases sympathetic nerve activity through promoting expression of proinflammatory cytokines in Sprague Dawley rats

doi: 10.1111/apha.12963

Figure Lengend Snippet: Orexin A treatment increases mRNA levels of PICs and Fra1 in PC12-OX1R cells. (A) Incubation of PC12-OX1R cells with orexin A for 6 hours resulted in a dose-dependent increase in mRNA levels of IL-6, TNF-α and Fra1 with maximum increase occurring in 100 nmol/L orexin A treatment. (B) Orexin A (100 nmol/L) stimulation caused time-dependent increase in mRNA level of IL-6, TNF-α and Fra1 in PC12-OX1R cells. (c) AP1 blocker curcumin (50 μmol L−1) attenuates the increase in the mRNA levels of PICs induced by orexin A. *P < .05, †P < .01, ††P < .001 vs Control

Article Snippet: Primary antibodies rabbit anti-OX1R and rabbit anti-OX2R were products of Alomone laboratories (Jerusalem, Israel); mouse anti-Fra1 antibody and mouse anti-TNFα antibody were from Santa Cruz Biotechnology.

Techniques: Incubation

Journal: Acta physiologica (Oxford, England)

Article Title: Orexin A increases sympathetic nerve activity through promoting expression of proinflammatory cytokines in Sprague Dawley rats

doi: 10.1111/apha.12963

Figure Lengend Snippet: Orexin A (100 nmol L−1) treatment increases immunoreactivity of Fra1 in PC12-OX1R cells. (A) The pictures show the DAPI staining (blue) and Fra1 staining (green) from cells in control and orexin A treatment group respectively. (B) The bar graph showing the summary data of statistical analysis of the Fra1 expression in cells from control and orexin A treatment group. ††P < .001 vs Control

Article Snippet: Primary antibodies rabbit anti-OX1R and rabbit anti-OX2R were products of Alomone laboratories (Jerusalem, Israel); mouse anti-Fra1 antibody and mouse anti-TNFα antibody were from Santa Cruz Biotechnology.

Techniques: Staining, Expressing

Journal: Acta physiologica (Oxford, England)

Article Title: Orexin A increases sympathetic nerve activity through promoting expression of proinflammatory cytokines in Sprague Dawley rats

doi: 10.1111/apha.12963

Figure Lengend Snippet: Orexin A (100 nmol L−1) treatment for 6 hours increases mRNA level of IL-6, TNF-α and Fra1 in PC12-OX1R, but not in PC12-OX2R and PC12 cells. *P < .05, ††P < .001 vs Control

Article Snippet: Primary antibodies rabbit anti-OX1R and rabbit anti-OX2R were products of Alomone laboratories (Jerusalem, Israel); mouse anti-Fra1 antibody and mouse anti-TNFα antibody were from Santa Cruz Biotechnology.

Techniques: