Journal: International Journal of Biological Sciences

Article Title: Activation of TLR3 Induces Osteogenic Responses in Human Aortic Valve Interstitial Cells through the NF-κB and ERK1/2 Pathways

doi: 10.7150/ijbs.10905

Figure Lengend Snippet: Poly(I:C) up-regulates the production of BMP-2, TGF-β1 and ALP in human AVICs in a dose-dependent fashion. AVICs isolated from normal human aortic valves were treated with varied concentrations of poly(I:C) for 24 hours. Representative immunoblots of three separated experiments and normalized densitometric data show that poly(I:C) in a dose range of 0.5-10 μg/mL increases BMP-2, TGF-β1 and ALP levels in human AVICs. While 0.5 μg/mL of poly(I:C) increases the levels of these osteogenic factors, 2.5 μg/mL results in maximal changes. No apparent further change is induced by poly(I:C) at a concentration greater than 2.5 μg/mL.

Article Snippet: Antibodies against BMP-2, TGF-β1, ALP, β-actin and TLR3 were purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA).

Techniques: Isolation, Western Blot, Concentration Assay

Journal: International Journal of Biological Sciences

Article Title: Activation of TLR3 Induces Osteogenic Responses in Human Aortic Valve Interstitial Cells through the NF-κB and ERK1/2 Pathways

doi: 10.7150/ijbs.10905

Figure Lengend Snippet: Poly(I:C) induces calcium deposits formation that is associated with elevated production of BMP-2, TGF-β1 and ALP. AVICs isolated from normal human aortic valves treated with 2.5 μg/mL poly(I:C) for varied period of time. A . Representative images and data from spectrophotometric analysis show that treatment of AVICs with poly(I:C) in conditioning medium (growth medium with 10 mmol/L of β-glycerophosphate, 10 nmol/L of vitamin D 3 , 10 nmol/L of dexamethasone, and 8 mmol/L of CaCl 2 ) for 10 days promotes the formation of calcium deposits. B. Representative immunoblots and normalized densitometric data show that treatment of AVICs with 2.5 μg/mL of poly(I:C) in normal growth medium for 24 hours up-regulates the production of BMP-2, TGF-β1 and ALP. Data are presented as mean ± standard error of 6 experiments using different AVIC isolates; * P <0.05 vs. untreated control (Ctrl).

Article Snippet: Antibodies against BMP-2, TGF-β1, ALP, β-actin and TLR3 were purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA).

Techniques: Isolation, Western Blot, Control

Journal: International Journal of Biological Sciences

Article Title: Activation of TLR3 Induces Osteogenic Responses in Human Aortic Valve Interstitial Cells through the NF-κB and ERK1/2 Pathways

doi: 10.7150/ijbs.10905

Figure Lengend Snippet: TLR3 mediates the up-regulation of BMP-2, TGF-β1 and ALP production by poly(I:C). AVICs were pre-treated with monoclonal TLR3-blocking antibody (clone TLR3.7, 20 μg/mL; non-immune mouse IgG for control) 1 hour before poly(I:C) stimulation or pre-treated with TLR3 siRNA (150 nmol/L; scrambled siRNA as control) 72 hours before poly(I:C) stimulation. Representative immunoblots and densitometric data show that blocking TLR3 with the antibody ( A ) and TLR3 knockdown ( B ) suppress the production of BMP-2, TGF-β1 and ALP induced by poly(I:C). Data are presented as mean ± standard error of 6 experiments using different AVIC isolates; * P <0.05 vs. untreated control (Ctrl); # P <0.05 vs. poly(I:C) alone; Δ P <0.05 vs. poly(I:C)+non-immune IgG; & P <0.05 vs. poly(I:C)+scrambled siRNA.

Article Snippet: Antibodies against BMP-2, TGF-β1, ALP, β-actin and TLR3 were purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA).

Techniques: Blocking Assay, Control, Western Blot, Knockdown

Journal: International Journal of Biological Sciences

Article Title: Activation of TLR3 Induces Osteogenic Responses in Human Aortic Valve Interstitial Cells through the NF-κB and ERK1/2 Pathways

doi: 10.7150/ijbs.10905

Figure Lengend Snippet: Both NF-κB and ERK1/2 are required for the up-regulation of the production of BMP-2, TGF-β1 and ALP by poly(I:C). AVICs were treated with IκB kinase inhibitor (Bay11-7082, 2.5 μmol/L) or a specific ERK1/2 inhibitor (328000ERK, 40 μmol/L) 1 hour prior to poly(I:C) stimulation for 24 hours. Representative immunoblots and densitometric data show that inhibition of either NF-κB ( A ) or ERK1/2 ( B ) abrogates the effect of poly(I:C) on BMP-2, TGF-β1 and ALP production. Data are presented as mean ± standard error of 6 experiments using different AVIC isolates; * P <0.05 vs. untreated control (Ctrl); # P <0.05 vs. poly(I:C) alone or poly(I:C)+DMSO.

Article Snippet: Antibodies against BMP-2, TGF-β1, ALP, β-actin and TLR3 were purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA).

Techniques: Western Blot, Inhibition, Control

Journal: Molecules and cells

Article Title: Transforming Growth Factor-β-Induced RBFOX3 Inhibition Promotes Epithelial-Mesenchymal Transition of Lung Cancer Cells.

doi: 10.14348/molcells.2016.0150

Figure Lengend Snippet: Fig. 3. Changes in the expression of EMT-related proteins following RBFOX3- depletion. (A) TGF-β1 treatment in- duced EMT in A549 cells. A549 cells were treated with TGF-β1 (5 ng/ml) for 48 h, and then cell lysates were sub- jected to immunoblot analysis for the indicated proteins. (B, C) Immunoblot analysis of EMT-related proteins. Con- struct of CRISPR-Cas9 for RBFOX3- KD were transfected into A549 cells. At 24 h after transfection, cells were treat- ed with TGF-β1 (5 ng/ml) for 48 h. The cell lysates were subjected to immunob- lot analysis for the indicated proteins. The values under the gels denote rela- tive intensities of the protein bands.

Article Snippet: Neutralizing anti-TGF-β antibody (1D11) and human TGF-β1 (R&D Systems, USA) were supplemented into the culture medium at concentrations of 25 μg/ml and 5 ng/ml, respectively.

Techniques: Expressing, Western Blot, CRISPR, Transfection

Journal: Molecules and cells

Article Title: Transforming Growth Factor-β-Induced RBFOX3 Inhibition Promotes Epithelial-Mesenchymal Transition of Lung Cancer Cells.

doi: 10.14348/molcells.2016.0150

Figure Lengend Snippet: Fig. 4. RBFOX3 depletion-induced morpho- logical changes in EMT phenotype. (A) RBFOX3-KD cells were treated with TGF- β1 (5 ng/ml) for 24 h. The fixed and perme- abilized cells were immunostained for the indicated proteins. Scale bars indicate 10 μm. (B) Quantification of NMHC II-A-stained cytoskeletal fibers. Each bar represents the number of NMHC II-A-stained cytoskeletal fibers in the cytoplasm (mean values ± SD, 10 cells). (C) Quantification of Vimentin- stained cytoskeletal fibers. Each bar repre- sents the intensity of Vimentin-stained cyto- skeletal fibers in the cytoplasm (mean val- ues ± SD, 10 cells). *P < 0.05 (two-sided t test) compared with control cells. (D) A model for the RBFOX3-mediated regulation of EMT processing. RBFOX3 expression is tran- scriptionally inhibited by TGF-β1. RBFOX3- inhibition stimulates morphological EMT phenotype.

Article Snippet: Neutralizing anti-TGF-β antibody (1D11) and human TGF-β1 (R&D Systems, USA) were supplemented into the culture medium at concentrations of 25 μg/ml and 5 ng/ml, respectively.

Techniques: Staining, Control, Expressing, Inhibition

Journal:

Article Title: Nerve growth factor displays stimulatory effects on human skin and lung fibroblasts, demonstrating a direct role for this factor in tissue repair

doi: 10.1073/pnas.101130898

Figure Lengend Snippet: Confocal photomicrographic analysis of trkA and p75 receptors. The basal expression of trkA by lung and skin fibroblasts (green) is shown in A and C, respectively. When cultured in the presence of 100 ng/ml NGF for 6 consecutive days, both lung and skin fibroblasts also expressed p75 (green, B and D, respectively). Red in the fibroblast nuclei is propidium iodide staining. Fibroblast monolayers incubated with nonspecific purified immunoglobulins (IgG) did not display positive staining. The experiment depicted is a representative one of three. (×40.)

Article Snippet: For the experiments, fibroblasts were used between the 3rd and 7th generation and cultured in medium (Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal calf serum, 2 mM l -glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin; Biological Industries, Beit Haemek, Israel) containing, according to the different experiments, one of the following: murine NGF (0.1–1000 ng/ml); purified human transforming growth factor β1 (TGF-β1; 20 ng/ml; R & D Systems); neutralizing goat anti-NGF antibodies, raised against ultrapure murine 2.5S NGF and further purified by column chromatography ( 16 ); or mouse anti-TGF-β1 monoclonal antibodies (10 μg/ml; R & D Systems).

Techniques: Expressing, Cell Culture, Staining, Incubation, Purification

Journal:

Article Title: Nerve growth factor displays stimulatory effects on human skin and lung fibroblasts, demonstrating a direct role for this factor in tissue repair

doi: 10.1073/pnas.101130898

Figure Lengend Snippet: Lung and skin fibroblast migration across a wound line as a function of NGF concentration. The effect of 50 ng/ml NGF on wounded lung and skin fibroblasts after 1 day is shown in A and B, respectively. (×5.) The experiment depicted is a representative one of three. (Bottom) Quantitative evaluation of fibroblast migration beyond the wound line (*, P < 0.05 for lung fibroblasts and **, P < 0.05 for skin fibroblasts, both at 50 and 100 ng/ml NGF). Experiments (n = 3) were performed in triplicates; error bars indicate SEM.

Article Snippet: For the experiments, fibroblasts were used between the 3rd and 7th generation and cultured in medium (Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal calf serum, 2 mM l -glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin; Biological Industries, Beit Haemek, Israel) containing, according to the different experiments, one of the following: murine NGF (0.1–1000 ng/ml); purified human transforming growth factor β1 (TGF-β1; 20 ng/ml; R & D Systems); neutralizing goat anti-NGF antibodies, raised against ultrapure murine 2.5S NGF and further purified by column chromatography ( 16 ); or mouse anti-TGF-β1 monoclonal antibodies (10 μg/ml; R & D Systems).

Techniques: Migration, Concentration Assay

Journal:

Article Title: Nerve growth factor displays stimulatory effects on human skin and lung fibroblasts, demonstrating a direct role for this factor in tissue repair

doi: 10.1073/pnas.101130898

Figure Lengend Snippet: Effect of NGF on α-SMA expression in lung and skin fibroblasts. Fibroblasts were cultured for 6 days with NGF or TGF-β1, and α-SMA expression by fibroblasts was evaluated by a cell surface ELISA. A and B show the effect of 100 ng/ml NGF and C and D, that of 10 ng/ml TGF-β1 on lung and skin fibroblasts, respectively. The experiment depicted is a representative one of three. (Bottom) Quantitative evaluation of α-SMA expression. α-SMA expression was found increased in both lung and skin fibroblasts (*, P < 0.05 for lung fibroblasts at 50 and 200 ng/ml NGF; **, P < 0.05 for skin fibroblasts at 50, 100, and 200 ng/ml NGF, and both for TGF-β1). Experiments (n = 3) were performed in triplicates; error bars indicate SEM.

Article Snippet: For the experiments, fibroblasts were used between the 3rd and 7th generation and cultured in medium (Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal calf serum, 2 mM l -glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin; Biological Industries, Beit Haemek, Israel) containing, according to the different experiments, one of the following: murine NGF (0.1–1000 ng/ml); purified human transforming growth factor β1 (TGF-β1; 20 ng/ml; R & D Systems); neutralizing goat anti-NGF antibodies, raised against ultrapure murine 2.5S NGF and further purified by column chromatography ( 16 ); or mouse anti-TGF-β1 monoclonal antibodies (10 μg/ml; R & D Systems).

Techniques: Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Journal of Clinical Medicine

Article Title: Analysis of Periostin, TGF-β, and SLUG Expression in Inflammatory Bowel Disease in Pediatric Patients and Their Clinical Implications

doi: 10.3390/jcm15020845

Figure Lengend Snippet: Statistical analysis of SNAI2 and TGF-β1 mRNA expression revealed that no significant differences in SNAI2 mRNA expression were observed between the IBD and control groups ( A ), whereas TGF-β1 mRNA expression was significantly higher in IBD compared with controls ( B ). Additionally, SNAI2 mRNA expression showed no significant differences between the control, CD, and UC groups ( C ), while TGF-β1 mRNA expression was higher in the CD and UC groups compared with controls ( D ). Furthermore, SNAI2 mRNA expression was higher in the ileum compared with the cecum, while in the remaining large-intestine segments there was only a non-significant trend toward higher SNAI2 expression in the ileum ( E ), and TGF-β1 mRNA expression was higher in the cecum compared with the other intestinal segments examined ( F ) (respectively: Mann–Whitney U test ( A , B ), Kruskal–Wallis test with Dunn’s post hoc test ( C – F ), * p < 0.05, ** p < 0.01). IBD—inflammatory bowel disease; CD—Crohn’s disease; UC—ulcerative colitis; TGF-β—transforming growth factor β; SNAI2—SNAIL family transcriptional repressor 2.

Article Snippet: The following primary antibodies were applied for 20 min at RT: periostin (1:100, NBP1–82472, Novus Biologicals, Littleton, CO, USA), TGF-β1 (1:100, NBP2–22114, Novus Biologicals), and SLUG (1:50, sc-166476, Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Expressing, Control, MANN-WHITNEY