Journal: Stem Cell Research & Therapy

Article Title: Evaluation of amniotic mesenchymal cell derivatives on cytokine production in equine alveolar macrophages: an in vitro approach to lung inflammation

doi: 10.1186/s13287-016-0398-9

Figure Lengend Snippet: Time-course and dose-response of lipopolysaccharide ( LPS )-induced cytokine production. Alveolar macrophages were seeded at 2 × 10 6 /mL and, after adherence, treated for 1, 24, 48, and 72 h in the presence or absence of LPS (10 and 100 ng/mL). The release of tumor necrosis factor alpha ( TNF - α ), interleukin-6 ( IL - 6 ), and transforming growth factor beta ( TGF - β ) were evaluated in conditioned medium by ELISA. Results are the mean of a minimum of four experiments ± SEM. Statistical analysis was performed by Tukey’s multiple comparison test, with * p < 0.05 versus control cells

Article Snippet: TNF-α, IL-6, and TGF-β1 measurements were made using commercially available ELISA kits (Genorise, Glen Mills, PA, USA) according to the manufacturer’s specifications.

Techniques: Enzyme-linked Immunosorbent Assay, Comparison, Control

Journal: Stem Cell Research & Therapy

Article Title: Evaluation of amniotic mesenchymal cell derivatives on cytokine production in equine alveolar macrophages: an in vitro approach to lung inflammation

doi: 10.1186/s13287-016-0398-9

Figure Lengend Snippet: Effects of CM and MVs on the production of pro- and anti-inflammatory cytokines. Alveolar macrophages were seeded at 2 × 10 6 /mL and, after adherence, treated for 1, 24, 48, and 72 h in the presence or absence of lipopolysaccharide ( LPS ) (10 and 100 ng/mL) or of conditioned medium ( cond. medium ) or microvesicles. The release of tumor necrosis factor alpha ( TNF - α ), interleukin-6 ( IL - 6 ), and transforming growth factor beta ( TGF - β ) were evaluated in conditioned medium by ELISA. Results are the mean of a minimum of three experiments ± SEM. Statistical analysis was performed by Tukey’s multiple comparison test, with § p < 0.05 vs LPS-treated cells

Article Snippet: TNF-α, IL-6, and TGF-β1 measurements were made using commercially available ELISA kits (Genorise, Glen Mills, PA, USA) according to the manufacturer’s specifications.

Techniques: Enzyme-linked Immunosorbent Assay, Comparison

Journal: Stem Cell Research & Therapy

Article Title: Evaluation of amniotic mesenchymal cell derivatives on cytokine production in equine alveolar macrophages: an in vitro approach to lung inflammation

doi: 10.1186/s13287-016-0398-9

Figure Lengend Snippet: Effect of transforming growth factor beta ( TGF - β ) and neutralizing anti-TGF-β antibody on lipopolysaccharide ( LPS )-induced tumor necrosis factor alpha ( TNF - α ) production. a Alveolar macrophages were seeded at 2 × 10 6 /mL and, after adherence, treated in the presence or absence of amniotic mesenchymal cell CM and MVs. Basal release of TGF-β was assessed by ELISA after 24 and 48 h of incubation. Each dot represents the value of a single animal. b After adherence, cells were treated in the presence or absence of equine TGF-β (300 pg/mL) and LPS (100 ng/mL) for 24 h. Cell viability was assessed by MTT test and TNF-α by ELISA. c After adherence, cells were treated in the presence or absence of anti-TGF-β antibody (0.2 μg/mL) or control mouse IgG (0.2 μg/mL) or CM and MVs, or LPS (100 ng/mL) for 24 h. TNF-α release was assessed by ELISA. Results are the mean of a minimum of three experiments ± SEM. Statistical analysis was performed by Tukey’s multiple comparison test, with ** p < 0.01 versus relative control cells and §§ p < 0.01 versus LPS-treated cells. OD optical density

Article Snippet: TNF-α, IL-6, and TGF-β1 measurements were made using commercially available ELISA kits (Genorise, Glen Mills, PA, USA) according to the manufacturer’s specifications.

Techniques: Enzyme-linked Immunosorbent Assay, Incubation, Control, Comparison

Journal: International heart journal

Article Title: Ryanodine Receptor Type 2 Plays a Role in the Development of Cardiac Fibrosis under Mechanical Stretch Through TGFβ-1.

doi: 10.1536/ihj.16-572

Figure Lengend Snippet: Figure 2. Reduction of TGFβ1 expression by downregulation of RyR2in cardiomyocytes under mechani- cal stretch. A: Measurement of RyR2 mRNA level in cultured cardiomyocytes by Real-Time PCR. B: Expres- sion of RyR2 protein level in cardiomyocytes with Western Blotting. C: TGFβ1 in supernates of cultured cardiomyocytes was detected with ELISA analysis. D: Expression of TGFβ1 protein level in cardiomyocytes with Western Blotting. E: Measurement of tgfb1 mRNA level in cultured cardiomyocytes using Real-Time PCR. Values are expressed as mean ± SEM obtained from 3 independent experiments. shRyR2 indicates RyR2 knockdown lentiviral particle-infected cardiomyocytes; MS, cardiomyocytes under mechanical stretch; MS + ryr2 KD, RyR2 knockdown cardiomyocytes under mechanical stretch. *P < 0.05 versus control.

Article Snippet: ELISA assay: Supernates of cultured cardiomyocytes were collected and examined by Rat Transforming Growth Factor β1 ELISA Kit (CUSABIO BIOTECH, China) following the manufacturer’s instructions.

Techniques: Expressing, Cell Culture, Real-time Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay, Knockdown, Infection, Control

Journal: PLoS ONE

Article Title: Smad2/3-Regulated Expression of DLX2 Is Associated with Radiation-Induced Epithelial-Mesenchymal Transition and Radioresistance of A549 and MDA-MB-231 Human Cancer Cell Lines

doi: 10.1371/journal.pone.0147343

Figure Lengend Snippet: (A) The levels of immunoreactive TGF-β1 were quantified from the cell culture medium by ELISA, as described in the Materials and methods (***P < 0.001, **P < 0.01, versus Ct). Ct, control cell; IR, irradiated cells. (B) Cells were transfected with 100 μM si-Smad2/3 or si-Ct, incubated at 37°C for 24 h. Then the cells were exposed to IR at 8Gy (A549) or 4Gy (MDA-MB-231) and incubated at 37°C for 24 h. Total RNA was isolated from the cells and subjected to real time PCR analysis. The graph represents the average of three independent experiments ±SE (***P < 0.001). (C) Cells were transfected with 100 μM si-Smad2/3 or si-Ct and incubated at 37°C for 24 h. Then the cells were exposed to IR at 8Gy (A549) or 4Gy (MDA-MB-231) and incubated at 37°C for 24 h. Subsequently, the cell lysates were subjected to western blot analysis. Three independent experiments obtained similar results. (D) Protein levels were quantified by densitometry. Data are represented as relative values to those of si-Ct after normalization with β-actin (***P < 0.001, **P < 0.01, *P < 0.05 versus si-Ct).

Article Snippet: A549 and MDA-MB-231 (5×10 5 cells/well) cells were exposed to IR at 8Gy, 4Gy and incubated at 37°C for 24 h. The level of TGF-β1 protein in culture supernatants was measured using a TGF-β1 ELISA kit (BD Biosciences, San Diego, USA) according to the manufacturer's instructions.

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Irradiation, Transfection, Incubation, Isolation, Real-time Polymerase Chain Reaction, Western Blot

Journal:

Article Title: Hepatitis C virus core protein promotes proliferation of human hepatoma cells through enhancement of transforming growth factor ? expression via activation of nuclear factor-?B

doi: 10.1136/gut.2005.070417

Figure Lengend Snippet: Figure 1 Enhancement of nuclear factor (NF)‐κB DNA‐binding activity by hepatitis C virus (HCV) core protein. (A) Schematic representation of the NF‐κB site in the transforming growth factor (TGF) α promoter region. We searched for the κB site in the promoter region of TGFα using a computer program and found an element with about 90% homogeneity to the consensus κB sequence between−215 and−206. (B) Electrophoretic mobility shift assay (EMSA) using NF‐κB binding sequence in the promoter region of TGFα. Huh‐7, HepG2 and Hep3B cells were transfected with the effector plasmid (pcDNA3/core) or the mock plasmid. After 24 h of transfection, nuclear extracts were prepared and assayed for the NF‐κB DNA‐binding activity by EMSA. Lane 1, 32P‐labelled free oligonucleotide; lane 2, nuclear extract prepared from cells transfected with mock plasmid; lane 3, nuclear extract prepared from cells transfected with pcDNA3/core; lane 4, competition assay (100‐fold of unlabelled oligonucleotide); lanes 5 and 6, supershift assay (lane 5, anti‐p50 antibody; lane 6, anti‐p65 antibody). Long arrows indicate DNA–NF‐κB complex. Short arrows indicate DNA–NF‐κB–antibody complex.

Article Snippet: Sandwich ELISA for TGFα was carried out using a TGFα ELISA kit (Oncogene Science, Uniondale, California, USA) according to the manufacturer's protocol.

Techniques: Binding Assay, Activity Assay, Virus, Sequencing, Electrophoretic Mobility Shift Assay, Transfection, Plasmid Preparation, Competitive Binding Assay

Journal:

Article Title: Hepatitis C virus core protein promotes proliferation of human hepatoma cells through enhancement of transforming growth factor ? expression via activation of nuclear factor-?B

doi: 10.1136/gut.2005.070417

Figure Lengend Snippet: Figure 2 (A) Phosphorylation and degradation of IκB in hepatoma cells transfected with hepatitis C virus (HCV) core plasmid. Huh‐7, HepG2 and Hep3B cells were transfected with the effector plasmid (pcDNA3/core) or mock plasmid. Whole cell lysates were collected 6 and 24 h after transfection and analysed by western blotting using anti‐IκB, anti‐phospho‐IκB and anti‐β‐actin antibodies. (B) Activation of transforming growth factor (TGF) α gene transcription through nuclear factor (NF)‐κB by HCV core protein. Huh‐7, HepG2 and Hep3B cells were transfected with 0.5 μg of pGL‐NFκB/TP (black column) or pGL‐NFκBm/TP (grey column) and an indicated amount of the effector plasmid (pcDNA3/core) or mock plasmid. After 24 h of transfection, luciferase activity was measured. It was normalised by assigning the activity of cells transfected with mock plasmid alone a value of 1 (relative luciferase activity). Error bars represent the mean (standard deviation) from three independent experiments. *p<0.05. c, core transfectant; m, mock transfectant; NT, no treatment.

Article Snippet: Sandwich ELISA for TGFα was carried out using a TGFα ELISA kit (Oncogene Science, Uniondale, California, USA) according to the manufacturer's protocol.

Techniques: Phospho-proteomics, Transfection, Virus, Plasmid Preparation, Western Blot, Activation Assay, Luciferase, Activity Assay, Standard Deviation

Journal:

Article Title: Hepatitis C virus core protein promotes proliferation of human hepatoma cells through enhancement of transforming growth factor ? expression via activation of nuclear factor-?B

doi: 10.1136/gut.2005.070417

Figure Lengend Snippet: Figure 3 Expression of transforming growth factor (TGF) α protein in hepatitis C virus (HCV) core‐expressing cells. Huh‐7, HepG2 and Hep3B cells were transfected with indicated amounts of the effector plasmid (pcDNA3/core) or mock plasmid. After 24 h of transfection, whole cell lysates were collected and analysed by western blotting. The β‐actin was used as an internal control. The lower panels show densitometric analyses of the bands. The values were normalised by assigning the intensity of cells transfected with mock plasmid alone a value of 1. Error bars represent the mean (standard deviation) from three independent experiments.

Article Snippet: Sandwich ELISA for TGFα was carried out using a TGFα ELISA kit (Oncogene Science, Uniondale, California, USA) according to the manufacturer's protocol.

Techniques: Expressing, Virus, Transfection, Plasmid Preparation, Western Blot, Control, Standard Deviation

Journal:

Article Title: Hepatitis C virus core protein promotes proliferation of human hepatoma cells through enhancement of transforming growth factor ? expression via activation of nuclear factor-?B

doi: 10.1136/gut.2005.070417

Figure Lengend Snippet: Figure 4 Secretion of transforming growth factor (TGF) α from hepatitis C virus (HCV) core‐expressing cells analysed by ELISA. Huh‐7 cells were transfected with indicated amounts of the effector plasmid (pcDNA3/core) or the mock plasmid. After 24 h of transfection, transforming growth factor (TGF) α concentrations in the culture media were measured by using a TGFα ELISA kit according to the manufacturer's protocol. Error bars represent the mean (standard deviation) from three independent experiments. *p<0.05; **p<0.01.

Article Snippet: Sandwich ELISA for TGFα was carried out using a TGFα ELISA kit (Oncogene Science, Uniondale, California, USA) according to the manufacturer's protocol.

Techniques: Virus, Expressing, Enzyme-linked Immunosorbent Assay, Transfection, Plasmid Preparation, Standard Deviation

Journal:

Article Title: Hepatitis C virus core protein promotes proliferation of human hepatoma cells through enhancement of transforming growth factor ? expression via activation of nuclear factor-?B

doi: 10.1136/gut.2005.070417

Figure Lengend Snippet: Figure 5 Inhibition of transforming growth factor (TGF) α transcription and protein expression by SN50 in hepatitis C virus (HCV) core‐expressing hepatoma cells. (A) Huh‐7, HepG2 and Hep3B cells were transfected with 0.5 μg of pGL‐nuclear factor (NF)‐κB/TP and indicated amounts of the effector plasmid (pcDNA3/core) or the mock plasmid and incubated for 24 h in the presence or absence of SN50, an inhibitor of nuclear translocation of NF‐κB or SN50M (mutated and inactive form) at a concentration of 50 μg/ml. *p<0.05. (B) Cells were transfected with 2 μg of the effector plasmid (pcDNA3/core) and incubated for 24 h in the presence or absence of SN50 or SN50M (50 μg/ml). After 24 h of transfection, whole cell lysates were collected and analysed by western blotting. The result shown is representative of three independent experiments. NT, no treatment.

Article Snippet: Sandwich ELISA for TGFα was carried out using a TGFα ELISA kit (Oncogene Science, Uniondale, California, USA) according to the manufacturer's protocol.

Techniques: Inhibition, Expressing, Virus, Transfection, Plasmid Preparation, Incubation, Translocation Assay, Concentration Assay, Western Blot

Journal:

Article Title: Hepatitis C virus core protein promotes proliferation of human hepatoma cells through enhancement of transforming growth factor ? expression via activation of nuclear factor-?B

doi: 10.1136/gut.2005.070417

Figure Lengend Snippet: Figure 7 Cell proliferation of hepatitis C virus (HCV) core‐transfected cells and inhibition of cell growth by SN50, anti‐transforming growth factor (TGF) α antibody and PD98059. (A) Huh‐7, HepG2 and Hep3B cells were transfected with pcDNA3/core (square symbols) or mock plasmid (circle symbols). A WST‐1 assay was carried out daily for 3 days and the relative absorbance was plotted. Mean (standard deviation) were determined from four independent experiments. At each time point, the statistical significance between the HCV core and mock transfectants were analysed by the two‐tailed Student's t test. *p<0.05 (B) Huh‐7 cells were transfected with the pcDNA3/core (open column) or the mock plasmid (filled column) and cultured for 24 h. Then, cells were further incubated for 24 h in the presence or absence of SN50 (50 μg/ml), SN50M (50 μg/ml), anti‐TGFα antibody (10 μg/ml) and PD98059 (10 μM). Cell proliferation was then analysed by the WST‐1 assay. Mean (SD) were determined from three independent experiments. *p<0.05.

Article Snippet: Sandwich ELISA for TGFα was carried out using a TGFα ELISA kit (Oncogene Science, Uniondale, California, USA) according to the manufacturer's protocol.

Techniques: Virus, Transfection, Inhibition, Plasmid Preparation, WST-1 Assay, Standard Deviation, Two Tailed Test, Cell Culture, Incubation