tgf β 1  (PeproTech)

 
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    Name:
    Recombinant Human TGF β1 CHO cell derived
    Description:
    The three mammalian isoforms of TGF β TGF β1 β2 β3 signal through the same receptor and elicit similar biological responses They are multifunctional cytokines that regulate cell proliferation growth differentiation and motility as well as synthesis and deposition of the extracellular matrix They are involved in various physiological processes including embryogenesis tissue remodeling and wound healing They are secreted predominantly as latent complexes which are stored at the cell surface and in the extracellular matrix The release of biologically active TGF β isoform from a latent complex involves proteolytic processing of the complex and or induction of conformational changes by proteins such as thrombospondin 1 TGF β1 is the most abundant isoform secreted by almost every cell type It was originally identified for its ability to induce phenotypic transformation of fibroblasts and recently it has been implicated in the formation of skin tumors Recombinant human TGF β1 is a 25 0 kDa protein composed of two identical 112 amino acid polypeptide chains linked by a single disulfide bond
    Catalog Number:
    100-21C-100UG
    Price:
    1,040.00
    Category:
    Recombinant Proteins
    Source:
    CHO cells
    Reactivity:
    Chicken Cow Dog Frog Monkey Mouse Pig Rabbit Rat
    Purity:
    98.0
    Quantity:
    100UG
    Buy from Supplier


    Structured Review

    PeproTech tgf β 1
    Puerarin inhibited <t>TGF-β1-induced</t> HUVECs migration rate . HUVECs were preincubated with different concentrations of puerarin (10, 25, and 50 μ M) for 30 min and then treated with TGF- <t>β</t> 1 (10 ng/ml) for 48 h until a monolayer was formed. Scratches were made straightly across the dishes. After rinsing twice, the medium was replaced by RPMI1640 with no FBS. Photographs were taken at indicated times (scale bars: 50 μ m).
    The three mammalian isoforms of TGF β TGF β1 β2 β3 signal through the same receptor and elicit similar biological responses They are multifunctional cytokines that regulate cell proliferation growth differentiation and motility as well as synthesis and deposition of the extracellular matrix They are involved in various physiological processes including embryogenesis tissue remodeling and wound healing They are secreted predominantly as latent complexes which are stored at the cell surface and in the extracellular matrix The release of biologically active TGF β isoform from a latent complex involves proteolytic processing of the complex and or induction of conformational changes by proteins such as thrombospondin 1 TGF β1 is the most abundant isoform secreted by almost every cell type It was originally identified for its ability to induce phenotypic transformation of fibroblasts and recently it has been implicated in the formation of skin tumors Recombinant human TGF β1 is a 25 0 kDa protein composed of two identical 112 amino acid polypeptide chains linked by a single disulfide bond
    https://www.bioz.com/result/tgf β 1/product/PeproTech
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    tgf β 1 - by Bioz Stars, 2021-05
    99/100 stars

    Images

    1) Product Images from "Puerarin Protects against Cardiac Fibrosis Associated with the Inhibition of TGF-β1/Smad2-Mediated Endothelial-to-Mesenchymal Transition"

    Article Title: Puerarin Protects against Cardiac Fibrosis Associated with the Inhibition of TGF-β1/Smad2-Mediated Endothelial-to-Mesenchymal Transition

    Journal: PPAR Research

    doi: 10.1155/2017/2647129

    Puerarin inhibited TGF-β1-induced HUVECs migration rate . HUVECs were preincubated with different concentrations of puerarin (10, 25, and 50 μ M) for 30 min and then treated with TGF- β 1 (10 ng/ml) for 48 h until a monolayer was formed. Scratches were made straightly across the dishes. After rinsing twice, the medium was replaced by RPMI1640 with no FBS. Photographs were taken at indicated times (scale bars: 50 μ m).
    Figure Legend Snippet: Puerarin inhibited TGF-β1-induced HUVECs migration rate . HUVECs were preincubated with different concentrations of puerarin (10, 25, and 50 μ M) for 30 min and then treated with TGF- β 1 (10 ng/ml) for 48 h until a monolayer was formed. Scratches were made straightly across the dishes. After rinsing twice, the medium was replaced by RPMI1640 with no FBS. Photographs were taken at indicated times (scale bars: 50 μ m).

    Techniques Used: Migration

    Puerarin inhibited Smad2 phosphorylation in HUVECs. HUVECs were preincubated with different concentrations of puerarin (10, 25, and 50 μ M) for 30 min and then treated with TGF- β 1 (10 ng/ml) for 48 h. Protein levels of p-Smad2 and Smad2 in cell lysates in indicated groups were detected by WB, normalized to GAPDH ( n = 6). ∗ p
    Figure Legend Snippet: Puerarin inhibited Smad2 phosphorylation in HUVECs. HUVECs were preincubated with different concentrations of puerarin (10, 25, and 50 μ M) for 30 min and then treated with TGF- β 1 (10 ng/ml) for 48 h. Protein levels of p-Smad2 and Smad2 in cell lysates in indicated groups were detected by WB, normalized to GAPDH ( n = 6). ∗ p

    Techniques Used: Western Blot

    GW9662 counteracted puerarin's suppression effect on EndMT. (a) HUVECs were preincubated with puerarin (50 μ M) or pioglitazone (20 μ M) in the presence or absence of GW9662 (10 μ M) for 30 min and then treated with TGF- β 1 (10 ng/ml) for 48 h. The protein levels of CD31, vimentin, p-Smad2, Smad2, Smad4, and PPAR- γ in cell lysates of indicated groups were detected by WB, normalized to GAPDH ( n = 6). ∗ p
    Figure Legend Snippet: GW9662 counteracted puerarin's suppression effect on EndMT. (a) HUVECs were preincubated with puerarin (50 μ M) or pioglitazone (20 μ M) in the presence or absence of GW9662 (10 μ M) for 30 min and then treated with TGF- β 1 (10 ng/ml) for 48 h. The protein levels of CD31, vimentin, p-Smad2, Smad2, Smad4, and PPAR- γ in cell lysates of indicated groups were detected by WB, normalized to GAPDH ( n = 6). ∗ p

    Techniques Used: Western Blot

    Puerarin inhibited TGF-β1-induced EndMT in HUVECs . (a–g) HUVECs were preincubated with different concentrations of puerarin (10, 25, and 50 μ M) for 30 min and then treated with TGF- β 1 (10 ng/ml) for 48 h. mRNA levels of CD31, vimentin, α -SMA, collagen I, collagen III, CTGF, and Fn in indicated groups were tested by RT-PCR, normalized to GAPDH ( n = 6). ∗ p
    Figure Legend Snippet: Puerarin inhibited TGF-β1-induced EndMT in HUVECs . (a–g) HUVECs were preincubated with different concentrations of puerarin (10, 25, and 50 μ M) for 30 min and then treated with TGF- β 1 (10 ng/ml) for 48 h. mRNA levels of CD31, vimentin, α -SMA, collagen I, collagen III, CTGF, and Fn in indicated groups were tested by RT-PCR, normalized to GAPDH ( n = 6). ∗ p

    Techniques Used: Reverse Transcription Polymerase Chain Reaction

    Pressure overload-induced cardiac fibrosis was alleviated in puerarin-treated mice . (a) Histological sections of the left ventricle in indicated groups were stained with PSR for fibrosis (upper and middle panel, scale bars: 100 μ m). α -SMA was detected with immunohistochemistry (lower panel, scale bars: 200 μ m). (b) TGF- β 1/Smad2 signaling pathway and α -SMA protein in indicated groups were determined by WB, normalized to GAPDH ( n = 6). ∗ p
    Figure Legend Snippet: Pressure overload-induced cardiac fibrosis was alleviated in puerarin-treated mice . (a) Histological sections of the left ventricle in indicated groups were stained with PSR for fibrosis (upper and middle panel, scale bars: 100 μ m). α -SMA was detected with immunohistochemistry (lower panel, scale bars: 200 μ m). (b) TGF- β 1/Smad2 signaling pathway and α -SMA protein in indicated groups were determined by WB, normalized to GAPDH ( n = 6). ∗ p

    Techniques Used: Mouse Assay, Staining, Immunohistochemistry, Western Blot

    2) Product Images from "Knockdown of FUT3 disrupts the proliferation, migration, tumorigenesis and TGF-β induced EMT in pancreatic cancer cells"

    Article Title: Knockdown of FUT3 disrupts the proliferation, migration, tumorigenesis and TGF-β induced EMT in pancreatic cancer cells

    Journal: Oncology Letters

    doi: 10.3892/ol.2018.8738

    Induction of EMT in pancreatic cancer cell by TGF-β is inhibited by knockdown of FUT3. (A) Cells treated with TGF-β exhibiting fibroblastic morphology (×400 magnification, red arrows). (B) Expression of marker genes for EMT were determined by western blot. FUT3, fucosyltransferases 3; TGF-β, transforming growth factor-β; EMT, epithelial-mesenchymal transition; SNAIL1, Snail Family Transcriptional Repressor 1; E-cadherin, epithelial cadherin.
    Figure Legend Snippet: Induction of EMT in pancreatic cancer cell by TGF-β is inhibited by knockdown of FUT3. (A) Cells treated with TGF-β exhibiting fibroblastic morphology (×400 magnification, red arrows). (B) Expression of marker genes for EMT were determined by western blot. FUT3, fucosyltransferases 3; TGF-β, transforming growth factor-β; EMT, epithelial-mesenchymal transition; SNAIL1, Snail Family Transcriptional Repressor 1; E-cadherin, epithelial cadherin.

    Techniques Used: Expressing, Marker, Western Blot

    3) Product Images from "Matrix stiffness regulates migration of human lung fibroblasts. Matrix stiffness regulates migration of human lung fibroblasts"

    Article Title: Matrix stiffness regulates migration of human lung fibroblasts. Matrix stiffness regulates migration of human lung fibroblasts

    Journal: Physiological Reports

    doi: 10.14814/phy2.13281

    Substrate stiffness regulates expression of α ‐ SMA and F‐actin. (A) Representative immunofluorescence images of lung fibroblasts cultured on increasing substrate stiffnesses with or without TGF ‐ β 1 (10 ng/mL) for 4 days, stained for α ‐ SMA (green), F‐actin (red), and nuclei (blue). Images were obtained using a confocal microscopy with a 25× objective. (B) Effects of substrate stiffness and TGF ‐ β 1 (10 ng/mL) on expression of α ‐ SMA proteins as assessed by Western blotting. (C) α ‐ SMA protein/ GAPDH protein ratios on different substrates without TGF ‐ β 1 treatment were compared ( n = 5). The α ‐ SMA / GAPDH ratio of the cells cultured on plastic dishes was defined as 1. Values are means ± SD . * P
    Figure Legend Snippet: Substrate stiffness regulates expression of α ‐ SMA and F‐actin. (A) Representative immunofluorescence images of lung fibroblasts cultured on increasing substrate stiffnesses with or without TGF ‐ β 1 (10 ng/mL) for 4 days, stained for α ‐ SMA (green), F‐actin (red), and nuclei (blue). Images were obtained using a confocal microscopy with a 25× objective. (B) Effects of substrate stiffness and TGF ‐ β 1 (10 ng/mL) on expression of α ‐ SMA proteins as assessed by Western blotting. (C) α ‐ SMA protein/ GAPDH protein ratios on different substrates without TGF ‐ β 1 treatment were compared ( n = 5). The α ‐ SMA / GAPDH ratio of the cells cultured on plastic dishes was defined as 1. Values are means ± SD . * P

    Techniques Used: Expressing, Immunofluorescence, Cell Culture, Staining, Confocal Microscopy, Western Blot

    Related Articles

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    Article Title: HMGA2 and Smads Co-regulate SNAIL1 Expression during Induction of Epithelial-to-Mesenchymal Transition *HMGA2 and Smads Co-regulate SNAIL1 Expression during Induction of Epithelial-to-Mesenchymal Transition * S⃞
    Article Snippet: .. Recombinant mature TGF-β1 was from PeproTech EC Ltd, London, UK. .. Cloning and Recombinant Protein Expression —The plasmid pcDNA3-HA-hHMGA2ΔC (amino acids 1–83) was cloned by PCR into the vector pcDNA3-HA C-terminally of the HA tag using the EcoRI and XhoI restriction sites.

    Article Title: Bcl11b Regulates IL-17 Through the TGF-β/Smad Pathway in HDM-Induced Asthma
    Article Snippet: To up-regulate Bcl11b expression in vitro , cells were transfected with 4 µg of mouse Bcl11b plasmid (GeneChem, Shanghai, China) using Amaxa Nucleofector Kit (Lonza, Basel, Switzerland). .. For TGF-β1 treatment, recombinant TGF-β1 (Peprotech, Rocky Hill, NJ, USA) was prepared as a stock solution at a concentration of 0.1 mg/mL in 10 mM citric acid. .. Cells were cultured with 5 µM TGF-β1 in the presence (pBcl11b + TGF-β1 group) or absence (TGF-β1 group) of pBcl11b.

    Article Title: Notch signaling is necessary for epithelial growth arrest by TGF-?
    Article Snippet: .. Recombinant mature TGF-β1 was purchased from PeproTech. ..

    Article Title: Tanshinone IIA Ameliorates CNS Autoimmunity by Promoting the Differentiation of Regulatory T Cells
    Article Snippet: Myelin oligodendrocyte glycoprotein (MOG)35–55 peptide (MEVGWYRSPFSRVVHLYRNGK, purity ≥ 98%) was purchased from GenScript (Nanjing, China). .. Recombinant murine granulocyte–macrophage colony-stimulating factor (rmGM-CSF), recombinant murine IL-4, and recombinant human transforming growth factor β1 (rhTGF-β1) were purchased from PeproTech (Rocky Hill, NJ). .. TGF-β-neutralizing monoclonal IgG1 (aTGF-β, clone: 1D11.16.8) was from Bio X Cell (West Lebanon, NH).

    Article Title: Glutaminolysis Promotes Collagen Translation and Stability via α-Ketoglutarate–mediated mTOR Activation and Proline Hydroxylation
    Article Snippet: .. Human recombinant transforming growth factor-β1 (TGF-β1) was obtained from PeproTech. ..

    Article Title: Tumor-associated macrophages promote tumor metastasis via the TGF-β/SOX9 axis in non-small cell lung cancer
    Article Snippet: .. In the experiments, A549 and H1299 cells were incubated with 10 ng/mL of recombinant human TGF-β (100-21C, PeproTech) in RPMI-1640 medium for 48 h. To stimulate transformation of THP-1 cells into macrophages, 50 ng/mL PMA (Sigma Chemical) were added to the medium for 48 h; cells were then washed three times with PBS and incubated for another 24 h in the absence of PMA. .. Co-culture procedures THP-1 derived macrophages and lung cancer cells were co-cultured using a cell culture insert (Corning, NY, USA) with a porous membrane to separate the upper and lower chambers.

    Article Title: CCN5 Reduces Ligamentum Flavum Hypertrophy by Modulating the TGF‐β Pathway
    Article Snippet: .. For the both cell types, exogenous recombinant human TGF‐β1 (Peprotech, Rocky Hill, NJ) was added to a 10 ng/ml final concentration in the TGF‐β1 groups, and an equal amount of PBS was added in the control groups. .. The exogeneous TGF‐β1 concentration was determined based on previously published and our Supplementary Data (Supplementary Fig. S1).

    Concentration Assay:

    Article Title: Bcl11b Regulates IL-17 Through the TGF-β/Smad Pathway in HDM-Induced Asthma
    Article Snippet: To up-regulate Bcl11b expression in vitro , cells were transfected with 4 µg of mouse Bcl11b plasmid (GeneChem, Shanghai, China) using Amaxa Nucleofector Kit (Lonza, Basel, Switzerland). .. For TGF-β1 treatment, recombinant TGF-β1 (Peprotech, Rocky Hill, NJ, USA) was prepared as a stock solution at a concentration of 0.1 mg/mL in 10 mM citric acid. .. Cells were cultured with 5 µM TGF-β1 in the presence (pBcl11b + TGF-β1 group) or absence (TGF-β1 group) of pBcl11b.

    Article Title: CCN5 Reduces Ligamentum Flavum Hypertrophy by Modulating the TGF‐β Pathway
    Article Snippet: .. For the both cell types, exogenous recombinant human TGF‐β1 (Peprotech, Rocky Hill, NJ) was added to a 10 ng/ml final concentration in the TGF‐β1 groups, and an equal amount of PBS was added in the control groups. .. The exogeneous TGF‐β1 concentration was determined based on previously published and our Supplementary Data (Supplementary Fig. S1).

    Incubation:

    Article Title: Tumor-associated macrophages promote tumor metastasis via the TGF-β/SOX9 axis in non-small cell lung cancer
    Article Snippet: .. In the experiments, A549 and H1299 cells were incubated with 10 ng/mL of recombinant human TGF-β (100-21C, PeproTech) in RPMI-1640 medium for 48 h. To stimulate transformation of THP-1 cells into macrophages, 50 ng/mL PMA (Sigma Chemical) were added to the medium for 48 h; cells were then washed three times with PBS and incubated for another 24 h in the absence of PMA. .. Co-culture procedures THP-1 derived macrophages and lung cancer cells were co-cultured using a cell culture insert (Corning, NY, USA) with a porous membrane to separate the upper and lower chambers.

    Transformation Assay:

    Article Title: Tumor-associated macrophages promote tumor metastasis via the TGF-β/SOX9 axis in non-small cell lung cancer
    Article Snippet: .. In the experiments, A549 and H1299 cells were incubated with 10 ng/mL of recombinant human TGF-β (100-21C, PeproTech) in RPMI-1640 medium for 48 h. To stimulate transformation of THP-1 cells into macrophages, 50 ng/mL PMA (Sigma Chemical) were added to the medium for 48 h; cells were then washed three times with PBS and incubated for another 24 h in the absence of PMA. .. Co-culture procedures THP-1 derived macrophages and lung cancer cells were co-cultured using a cell culture insert (Corning, NY, USA) with a porous membrane to separate the upper and lower chambers.

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  • 99
    PeproTech tgf β 1
    Puerarin inhibited <t>TGF-β1-induced</t> HUVECs migration rate . HUVECs were preincubated with different concentrations of puerarin (10, 25, and 50 μ M) for 30 min and then treated with TGF- <t>β</t> 1 (10 ng/ml) for 48 h until a monolayer was formed. Scratches were made straightly across the dishes. After rinsing twice, the medium was replaced by RPMI1640 with no FBS. Photographs were taken at indicated times (scale bars: 50 μ m).
    Tgf β 1, supplied by PeproTech, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/tgf β 1/product/PeproTech
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    tgf β 1 - by Bioz Stars, 2021-05
    99/100 stars
      Buy from Supplier

    96
    PeproTech recombinant human tgf β
    Dedifferentiation occurs during blocking of fibrosis by eupatilin. ONGHEPA1 cells were stimulated with <t>TGF-β</t> for 72 h or not stimulated; in parallel, another set of ONGHEPA1 cells were stimulated with TGF-β for 24 h, washed, and stimulated with TGF-β + eupatilin for 48 h. After washing, the resultant cells were continuously stimulated with TGF-β for 72 h. Morphological changes were monitored under a light microscope. Each pathological stage is indicated by arrows and corresponding terms.
    Recombinant Human Tgf β, supplied by PeproTech, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/recombinant human tgf β/product/PeproTech
    Average 96 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    recombinant human tgf β - by Bioz Stars, 2021-05
    96/100 stars
      Buy from Supplier

    93
    PeproTech tgfβ2
    Differentially expression of canonical TGFβ/BMP-SMAD genes in ERG-deficient HUVEC. a Microarray analysis of ERG-dependent genes in HUVEC was performed at 24 and 48 h after ERG depletion, as described ( n = 3 biological replicates) 24 ; fold change (log 2) of selected TGFβ/BMP associated genes represented as high (red) and low (blue) expression compared to the median (white). Gene expression data were validated in HUVEC transfected with Control (Con siRNA) or ERG siRNA by b quantitative PCR and c immunoblotting, showing reduction of ERG protein levels following siRNA, d quantification by fluorescence intensity normalised to GAPDH for ERG, SMAD1, ENG, ID1, SMAD3, ALK5 and SMA ( n = 5). e Representative images of SMAD1 expression (white; arrows identify expression in the sinusoidal endothelium), VWF (green), SMA (red) and DAPI (blue) in liver tissue from Erg fl/fl and Erg cEC-het mice (Scale bar 50 μm). f Quantitative analysis of <t>TGFβ2</t> protein expression was performed by ELISA in whole cell HUVEC lysates ( n = 7). g Representative image of TGFβ2 expression in HUVEC transfected with control or ERG siRNA by immunofluorescence; nuclei are identified by DRAQ V and cells are co-stained for ERG (green). Scale bar 20 µm. h Representative images of TGFβ2 expression (white; arrows) in large vessel within the liver from Erg fl/fl and Erg cEC-het mice (Scale bar 50 μm). Data were normalised to GAPDH and compared to control siRNA treated (*) by unpaired t -test. All graphical data are mean ± s.e.m., * P
    Tgfβ2, supplied by PeproTech, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/tgfβ2/product/PeproTech
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    tgfβ2 - by Bioz Stars, 2021-05
    93/100 stars
      Buy from Supplier

    93
    PeproTech human tgf β
    Dectin-1-activated DCs enhance Th9 cell differentiation in vitro . Naive CD4 + T cells from spleens of mice ( n =3–5) were cocultured with DCs matured with TNF-α/IL-1β (BMDC), Curlan (CurDC) or Scleroglucan (SclDC) in the presence of anti-CD3 with (Th9) or without (Th0) addition of Th9-polarizing cytokines <t>TGF-β</t> and IL-4 for 3 days. Culture supernatants and CD4 + T cells separated by the magnetic cell sorting (MACS) were collected for analysis. ( a ) Cells were stained with anti-CD4 and anti-IL-9 antibodies and subjected to flow cytometry analysis. Numbers in the dot plots represent the percentages of CD4 + IL-9 + T cells. Right, summarized results of four independent experiments obtained as at left. ( b ) ELISA assessed the IL-9 secretion in the cocultures. ( c , d ) qPCR analysis of Th9-, Th1-, Th2- and Th17-related cytokines ( c ) and transcription factors ( d ). Expression was normalized to Gapdh and set at 1 in BMDC-induced Th9 cells. Results shown are the mean±s.d. of 3–5 independent experiments. * P
    Human Tgf β, supplied by PeproTech, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/human tgf β/product/PeproTech
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    human tgf β - by Bioz Stars, 2021-05
    93/100 stars
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    Image Search Results


    Puerarin inhibited TGF-β1-induced HUVECs migration rate . HUVECs were preincubated with different concentrations of puerarin (10, 25, and 50 μ M) for 30 min and then treated with TGF- β 1 (10 ng/ml) for 48 h until a monolayer was formed. Scratches were made straightly across the dishes. After rinsing twice, the medium was replaced by RPMI1640 with no FBS. Photographs were taken at indicated times (scale bars: 50 μ m).

    Journal: PPAR Research

    Article Title: Puerarin Protects against Cardiac Fibrosis Associated with the Inhibition of TGF-β1/Smad2-Mediated Endothelial-to-Mesenchymal Transition

    doi: 10.1155/2017/2647129

    Figure Lengend Snippet: Puerarin inhibited TGF-β1-induced HUVECs migration rate . HUVECs were preincubated with different concentrations of puerarin (10, 25, and 50 μ M) for 30 min and then treated with TGF- β 1 (10 ng/ml) for 48 h until a monolayer was formed. Scratches were made straightly across the dishes. After rinsing twice, the medium was replaced by RPMI1640 with no FBS. Photographs were taken at indicated times (scale bars: 50 μ m).

    Article Snippet: TGF-β 1 was purchased from PEPROTECH (100-21C).

    Techniques: Migration

    Puerarin inhibited Smad2 phosphorylation in HUVECs. HUVECs were preincubated with different concentrations of puerarin (10, 25, and 50 μ M) for 30 min and then treated with TGF- β 1 (10 ng/ml) for 48 h. Protein levels of p-Smad2 and Smad2 in cell lysates in indicated groups were detected by WB, normalized to GAPDH ( n = 6). ∗ p

    Journal: PPAR Research

    Article Title: Puerarin Protects against Cardiac Fibrosis Associated with the Inhibition of TGF-β1/Smad2-Mediated Endothelial-to-Mesenchymal Transition

    doi: 10.1155/2017/2647129

    Figure Lengend Snippet: Puerarin inhibited Smad2 phosphorylation in HUVECs. HUVECs were preincubated with different concentrations of puerarin (10, 25, and 50 μ M) for 30 min and then treated with TGF- β 1 (10 ng/ml) for 48 h. Protein levels of p-Smad2 and Smad2 in cell lysates in indicated groups were detected by WB, normalized to GAPDH ( n = 6). ∗ p

    Article Snippet: TGF-β 1 was purchased from PEPROTECH (100-21C).

    Techniques: Western Blot

    GW9662 counteracted puerarin's suppression effect on EndMT. (a) HUVECs were preincubated with puerarin (50 μ M) or pioglitazone (20 μ M) in the presence or absence of GW9662 (10 μ M) for 30 min and then treated with TGF- β 1 (10 ng/ml) for 48 h. The protein levels of CD31, vimentin, p-Smad2, Smad2, Smad4, and PPAR- γ in cell lysates of indicated groups were detected by WB, normalized to GAPDH ( n = 6). ∗ p

    Journal: PPAR Research

    Article Title: Puerarin Protects against Cardiac Fibrosis Associated with the Inhibition of TGF-β1/Smad2-Mediated Endothelial-to-Mesenchymal Transition

    doi: 10.1155/2017/2647129

    Figure Lengend Snippet: GW9662 counteracted puerarin's suppression effect on EndMT. (a) HUVECs were preincubated with puerarin (50 μ M) or pioglitazone (20 μ M) in the presence or absence of GW9662 (10 μ M) for 30 min and then treated with TGF- β 1 (10 ng/ml) for 48 h. The protein levels of CD31, vimentin, p-Smad2, Smad2, Smad4, and PPAR- γ in cell lysates of indicated groups were detected by WB, normalized to GAPDH ( n = 6). ∗ p

    Article Snippet: TGF-β 1 was purchased from PEPROTECH (100-21C).

    Techniques: Western Blot

    Puerarin inhibited TGF-β1-induced EndMT in HUVECs . (a–g) HUVECs were preincubated with different concentrations of puerarin (10, 25, and 50 μ M) for 30 min and then treated with TGF- β 1 (10 ng/ml) for 48 h. mRNA levels of CD31, vimentin, α -SMA, collagen I, collagen III, CTGF, and Fn in indicated groups were tested by RT-PCR, normalized to GAPDH ( n = 6). ∗ p

    Journal: PPAR Research

    Article Title: Puerarin Protects against Cardiac Fibrosis Associated with the Inhibition of TGF-β1/Smad2-Mediated Endothelial-to-Mesenchymal Transition

    doi: 10.1155/2017/2647129

    Figure Lengend Snippet: Puerarin inhibited TGF-β1-induced EndMT in HUVECs . (a–g) HUVECs were preincubated with different concentrations of puerarin (10, 25, and 50 μ M) for 30 min and then treated with TGF- β 1 (10 ng/ml) for 48 h. mRNA levels of CD31, vimentin, α -SMA, collagen I, collagen III, CTGF, and Fn in indicated groups were tested by RT-PCR, normalized to GAPDH ( n = 6). ∗ p

    Article Snippet: TGF-β 1 was purchased from PEPROTECH (100-21C).

    Techniques: Reverse Transcription Polymerase Chain Reaction

    Pressure overload-induced cardiac fibrosis was alleviated in puerarin-treated mice . (a) Histological sections of the left ventricle in indicated groups were stained with PSR for fibrosis (upper and middle panel, scale bars: 100 μ m). α -SMA was detected with immunohistochemistry (lower panel, scale bars: 200 μ m). (b) TGF- β 1/Smad2 signaling pathway and α -SMA protein in indicated groups were determined by WB, normalized to GAPDH ( n = 6). ∗ p

    Journal: PPAR Research

    Article Title: Puerarin Protects against Cardiac Fibrosis Associated with the Inhibition of TGF-β1/Smad2-Mediated Endothelial-to-Mesenchymal Transition

    doi: 10.1155/2017/2647129

    Figure Lengend Snippet: Pressure overload-induced cardiac fibrosis was alleviated in puerarin-treated mice . (a) Histological sections of the left ventricle in indicated groups were stained with PSR for fibrosis (upper and middle panel, scale bars: 100 μ m). α -SMA was detected with immunohistochemistry (lower panel, scale bars: 200 μ m). (b) TGF- β 1/Smad2 signaling pathway and α -SMA protein in indicated groups were determined by WB, normalized to GAPDH ( n = 6). ∗ p

    Article Snippet: TGF-β 1 was purchased from PEPROTECH (100-21C).

    Techniques: Mouse Assay, Staining, Immunohistochemistry, Western Blot

    Dedifferentiation occurs during blocking of fibrosis by eupatilin. ONGHEPA1 cells were stimulated with TGF-β for 72 h or not stimulated; in parallel, another set of ONGHEPA1 cells were stimulated with TGF-β for 24 h, washed, and stimulated with TGF-β + eupatilin for 48 h. After washing, the resultant cells were continuously stimulated with TGF-β for 72 h. Morphological changes were monitored under a light microscope. Each pathological stage is indicated by arrows and corresponding terms.

    Journal: EBioMedicine

    Article Title: Reversion of in vivo fibrogenesis by novel chromone scaffolds

    doi: 10.1016/j.ebiom.2018.12.017

    Figure Lengend Snippet: Dedifferentiation occurs during blocking of fibrosis by eupatilin. ONGHEPA1 cells were stimulated with TGF-β for 72 h or not stimulated; in parallel, another set of ONGHEPA1 cells were stimulated with TGF-β for 24 h, washed, and stimulated with TGF-β + eupatilin for 48 h. After washing, the resultant cells were continuously stimulated with TGF-β for 72 h. Morphological changes were monitored under a light microscope. Each pathological stage is indicated by arrows and corresponding terms.

    Article Snippet: Recombinant human TGF-β and PDGF were obtained from Peprotech (Rocky Hill, CT, USA) and used at a final concentration of 5 ng/ml.

    Techniques: Blocking Assay, Light Microscopy

    Structure–activity relationship of a selected group of chromone derivatives. (a) Chemical structure of the general chromone scaffold, with its carbons denoted in colored fonts. C2, C3, C6, and C7 are critical for anti-fibrogenesis. (b) ONGHEPA1 cells were simultaneously stimulated with control medium, TGF-β, or TGF-β plus eupatilin (50 μM). Anti-fibrogenic effects were observed by phase-contrast light microscopy. (c) A list of 35 CDs, grouped by chemical moieties coupled to the chromone scaffolds or the phenyl ring. Group A, including eupatilin, ONGA300 (jaceosidin), and ONGH300 (hispidulin), exerted potent anti-fibrogenic capacity. (d) Anti-fibrogenic effects of ONGH300, observed by phase-contrast light microscopy. (e) Anti-fibrogenic effects of ONGI200 (also called apigenin), observed by phase-contrast light microscopy.

    Journal: EBioMedicine

    Article Title: Reversion of in vivo fibrogenesis by novel chromone scaffolds

    doi: 10.1016/j.ebiom.2018.12.017

    Figure Lengend Snippet: Structure–activity relationship of a selected group of chromone derivatives. (a) Chemical structure of the general chromone scaffold, with its carbons denoted in colored fonts. C2, C3, C6, and C7 are critical for anti-fibrogenesis. (b) ONGHEPA1 cells were simultaneously stimulated with control medium, TGF-β, or TGF-β plus eupatilin (50 μM). Anti-fibrogenic effects were observed by phase-contrast light microscopy. (c) A list of 35 CDs, grouped by chemical moieties coupled to the chromone scaffolds or the phenyl ring. Group A, including eupatilin, ONGA300 (jaceosidin), and ONGH300 (hispidulin), exerted potent anti-fibrogenic capacity. (d) Anti-fibrogenic effects of ONGH300, observed by phase-contrast light microscopy. (e) Anti-fibrogenic effects of ONGI200 (also called apigenin), observed by phase-contrast light microscopy.

    Article Snippet: Recombinant human TGF-β and PDGF were obtained from Peprotech (Rocky Hill, CT, USA) and used at a final concentration of 5 ng/ml.

    Techniques: Activity Assay, Light Microscopy

    Eupatilin inhibits TGF-β–mediated cellular response by dismantling latent TGF-β complex, depolymerizing actin, and modulating Smad3. (a) ONGHEPA1 cells were stimulated with TGF-β (5 ng/ml) or TGF-β plus eupatilin (50 μM) for 24 h, and morphological changes were monitored under a light microscope. ICC was performed using anti-mouse αSMA antibody. Total RNAs were isolated from ONGHEPA1 cells stimulated with medium, TGF-β, or TGF-β plus eupatilin for 48 h, and then subjected to real-time PCR analysis using primers against Col11a1 . (b) ONGHEPA1 cells were grown and stimulated with medium, apigenin, eupatilin, pirfenidone, or nintedanib in the presence or absence of TGF-β for 24 h, and then anti-fibrogenic activity was monitored. (c) Kinetics of eupatilin-induced Erk, Smad2, Smad3, and Akt phosphorylation in the presence of TGF-β. Western blot analyses of pErk1/2, pSmad2, pSmad3, and pAkt levels were performed in whole-cell lysates (30 μg) of ONGHEPA1 cells incubated with TGF-β or TGF-β and eupatilin for 0, 5, 15, 30, 45, 60, and 120 min. (d) Actin depolymerization and latent TGF-β complex–associated protein expression in eupatilin, pirfenidone, and nintedanib-treated ONGHEPA1 cells in the presence of TGF-β. ONGHEPA1 cells stained for F-actin (phalloidin, red), mLTBP (green), mLAP1 (green), and DAPI (nuclei, blue). Only eupatilin was able to depolymerize actin.

    Journal: EBioMedicine

    Article Title: Reversion of in vivo fibrogenesis by novel chromone scaffolds

    doi: 10.1016/j.ebiom.2018.12.017

    Figure Lengend Snippet: Eupatilin inhibits TGF-β–mediated cellular response by dismantling latent TGF-β complex, depolymerizing actin, and modulating Smad3. (a) ONGHEPA1 cells were stimulated with TGF-β (5 ng/ml) or TGF-β plus eupatilin (50 μM) for 24 h, and morphological changes were monitored under a light microscope. ICC was performed using anti-mouse αSMA antibody. Total RNAs were isolated from ONGHEPA1 cells stimulated with medium, TGF-β, or TGF-β plus eupatilin for 48 h, and then subjected to real-time PCR analysis using primers against Col11a1 . (b) ONGHEPA1 cells were grown and stimulated with medium, apigenin, eupatilin, pirfenidone, or nintedanib in the presence or absence of TGF-β for 24 h, and then anti-fibrogenic activity was monitored. (c) Kinetics of eupatilin-induced Erk, Smad2, Smad3, and Akt phosphorylation in the presence of TGF-β. Western blot analyses of pErk1/2, pSmad2, pSmad3, and pAkt levels were performed in whole-cell lysates (30 μg) of ONGHEPA1 cells incubated with TGF-β or TGF-β and eupatilin for 0, 5, 15, 30, 45, 60, and 120 min. (d) Actin depolymerization and latent TGF-β complex–associated protein expression in eupatilin, pirfenidone, and nintedanib-treated ONGHEPA1 cells in the presence of TGF-β. ONGHEPA1 cells stained for F-actin (phalloidin, red), mLTBP (green), mLAP1 (green), and DAPI (nuclei, blue). Only eupatilin was able to depolymerize actin.

    Article Snippet: Recombinant human TGF-β and PDGF were obtained from Peprotech (Rocky Hill, CT, USA) and used at a final concentration of 5 ng/ml.

    Techniques: Light Microscopy, Immunocytochemistry, Isolation, Real-time Polymerase Chain Reaction, Activity Assay, Western Blot, Incubation, Expressing, Staining

    Inhibition of TGF-β–induced fibrosis by eupatilin. (a) DHLFs (5 × 10 6 cells) were seeded in fibroblast growth medium (FBM), grown overnight, and then stimulated with control medium, TGF-β (5 ng/ml), TGF-β plus eupatilin (50 μM), or TGF-β plus pirfenidone (50 μM) for 72 h. Morphological change was monitored by phase-contrast microscopy at 40× magnification. (b) Proliferation of DHLFs was substantially attenuated by eupatilin and pirfenidone. DHLFs were treated with 0, 25, 50, and 100 μM eupatilin or pirfenidone in the presence of TGF-β for the indicated periods prior to estimation of cell proliferation using the CCK-8 assay. The experiment was performed in triplicate. (c) Eupatilin, but not pirfenidone, inhibited TGF-β–induced α–smooth muscle actin (αSMA). ICC was performed using anti-mouse αSMA antibody. (d) Total RNA was isolated from DHLFs stimulated with control medium, TGF-β, TGF-β plus eupatilin, or TGF-β plus pirfenidone for 48 h, and then subjected to real-time PCR analysis using primers against POSTN or VIM . Standard deviations were calculated from the results of three independent PCRs.

    Journal: EBioMedicine

    Article Title: Reversion of in vivo fibrogenesis by novel chromone scaffolds

    doi: 10.1016/j.ebiom.2018.12.017

    Figure Lengend Snippet: Inhibition of TGF-β–induced fibrosis by eupatilin. (a) DHLFs (5 × 10 6 cells) were seeded in fibroblast growth medium (FBM), grown overnight, and then stimulated with control medium, TGF-β (5 ng/ml), TGF-β plus eupatilin (50 μM), or TGF-β plus pirfenidone (50 μM) for 72 h. Morphological change was monitored by phase-contrast microscopy at 40× magnification. (b) Proliferation of DHLFs was substantially attenuated by eupatilin and pirfenidone. DHLFs were treated with 0, 25, 50, and 100 μM eupatilin or pirfenidone in the presence of TGF-β for the indicated periods prior to estimation of cell proliferation using the CCK-8 assay. The experiment was performed in triplicate. (c) Eupatilin, but not pirfenidone, inhibited TGF-β–induced α–smooth muscle actin (αSMA). ICC was performed using anti-mouse αSMA antibody. (d) Total RNA was isolated from DHLFs stimulated with control medium, TGF-β, TGF-β plus eupatilin, or TGF-β plus pirfenidone for 48 h, and then subjected to real-time PCR analysis using primers against POSTN or VIM . Standard deviations were calculated from the results of three independent PCRs.

    Article Snippet: Recombinant human TGF-β and PDGF were obtained from Peprotech (Rocky Hill, CT, USA) and used at a final concentration of 5 ng/ml.

    Techniques: Inhibition, Microscopy, CCK-8 Assay, Immunocytochemistry, Isolation, Real-time Polymerase Chain Reaction

    Dedifferentiation is associated with eupatilin-mediated anti-fibrotic capacity. (a and c) ONGHEPA1 cells were stimulated with TGF-β or PDGF for 48 h, and another set of ONGHEPA1 cells was first stimulated with TGF-β or PDGF for 24 h until myofibroblasts were predominant, and then further stimulated with both TGF-β + eupatilin or TGF-β + ONGE200 and PDGF + eupatilin or PDGF + ONGE200. Morphological changes were monitored under a light microscope. (b and d) Total RNA was isolated from ONGHEPA1 cells stimulated as described above and subjected to real-time PCR analysis using primers against Postn or Fn1 . Standard deviations were calculated from the results of three independent PCRs.

    Journal: EBioMedicine

    Article Title: Reversion of in vivo fibrogenesis by novel chromone scaffolds

    doi: 10.1016/j.ebiom.2018.12.017

    Figure Lengend Snippet: Dedifferentiation is associated with eupatilin-mediated anti-fibrotic capacity. (a and c) ONGHEPA1 cells were stimulated with TGF-β or PDGF for 48 h, and another set of ONGHEPA1 cells was first stimulated with TGF-β or PDGF for 24 h until myofibroblasts were predominant, and then further stimulated with both TGF-β + eupatilin or TGF-β + ONGE200 and PDGF + eupatilin or PDGF + ONGE200. Morphological changes were monitored under a light microscope. (b and d) Total RNA was isolated from ONGHEPA1 cells stimulated as described above and subjected to real-time PCR analysis using primers against Postn or Fn1 . Standard deviations were calculated from the results of three independent PCRs.

    Article Snippet: Recombinant human TGF-β and PDGF were obtained from Peprotech (Rocky Hill, CT, USA) and used at a final concentration of 5 ng/ml.

    Techniques: Light Microscopy, Isolation, Real-time Polymerase Chain Reaction

    Proposed MOAs of eupatilin, leading to anti-fibrotic capacity. Eupatilin directly acts on pathogenic myofibroblasts and inhibits fibrosis. Eupatilin rapidly depolymerizes actin stress fibers to which latent TGF-β complex is anchored. Under pathologic circumstances, the TGF-β cargo proteome, consisting of a heterodimeric integrin (mainly integrin avβ3 ) loaded with RGD, LAP1, and LTBP1, carry on the processed and active TGF-β and is relayed to TGFR. LTBP1 is secreted into the ECM. Upon actin depolymerization, latent TGF-β complex is dismantled, leaving active TGF-β at the cargo proteome and nullifying its binding to TGFR1. By dephosphorylation of Smad3 induction of EMT master regulators such as Snail2 may not be made possible. Eupatilin causes myofibroblasts to revert to HSC-like intermediate cell types. Whether actin depolymerization is responsible for this dedifferentiation remains to be scrutinized. Finally, eupatilin, like pirfenidone, inhibits in vivo TGF-β production.

    Journal: EBioMedicine

    Article Title: Reversion of in vivo fibrogenesis by novel chromone scaffolds

    doi: 10.1016/j.ebiom.2018.12.017

    Figure Lengend Snippet: Proposed MOAs of eupatilin, leading to anti-fibrotic capacity. Eupatilin directly acts on pathogenic myofibroblasts and inhibits fibrosis. Eupatilin rapidly depolymerizes actin stress fibers to which latent TGF-β complex is anchored. Under pathologic circumstances, the TGF-β cargo proteome, consisting of a heterodimeric integrin (mainly integrin avβ3 ) loaded with RGD, LAP1, and LTBP1, carry on the processed and active TGF-β and is relayed to TGFR. LTBP1 is secreted into the ECM. Upon actin depolymerization, latent TGF-β complex is dismantled, leaving active TGF-β at the cargo proteome and nullifying its binding to TGFR1. By dephosphorylation of Smad3 induction of EMT master regulators such as Snail2 may not be made possible. Eupatilin causes myofibroblasts to revert to HSC-like intermediate cell types. Whether actin depolymerization is responsible for this dedifferentiation remains to be scrutinized. Finally, eupatilin, like pirfenidone, inhibits in vivo TGF-β production.

    Article Snippet: Recombinant human TGF-β and PDGF were obtained from Peprotech (Rocky Hill, CT, USA) and used at a final concentration of 5 ng/ml.

    Techniques: Binding Assay, De-Phosphorylation Assay, In Vivo

    The interactome generated by simultaneous stimulation of ONGHEPA1 cells with TGF-β and eupatilin reveals a transdifferentiation and/or dedifferentiation pathway. Total RNA was isolated from ONGHEPA1 cells treated with TGF-β or TGF-β plus eupatilin. One microgram of RNA from each sample was subjected to RNA-seq, yielding 60 Gb of sequence, and gene interactions were established as described in Materials and Methods. Threshold for selection of a gene as differentially regulated was p

    Journal: EBioMedicine

    Article Title: Reversion of in vivo fibrogenesis by novel chromone scaffolds

    doi: 10.1016/j.ebiom.2018.12.017

    Figure Lengend Snippet: The interactome generated by simultaneous stimulation of ONGHEPA1 cells with TGF-β and eupatilin reveals a transdifferentiation and/or dedifferentiation pathway. Total RNA was isolated from ONGHEPA1 cells treated with TGF-β or TGF-β plus eupatilin. One microgram of RNA from each sample was subjected to RNA-seq, yielding 60 Gb of sequence, and gene interactions were established as described in Materials and Methods. Threshold for selection of a gene as differentially regulated was p

    Article Snippet: Recombinant human TGF-β and PDGF were obtained from Peprotech (Rocky Hill, CT, USA) and used at a final concentration of 5 ng/ml.

    Techniques: Generated, Isolation, RNA Sequencing Assay, Sequencing, Selection

    Differentially expression of canonical TGFβ/BMP-SMAD genes in ERG-deficient HUVEC. a Microarray analysis of ERG-dependent genes in HUVEC was performed at 24 and 48 h after ERG depletion, as described ( n = 3 biological replicates) 24 ; fold change (log 2) of selected TGFβ/BMP associated genes represented as high (red) and low (blue) expression compared to the median (white). Gene expression data were validated in HUVEC transfected with Control (Con siRNA) or ERG siRNA by b quantitative PCR and c immunoblotting, showing reduction of ERG protein levels following siRNA, d quantification by fluorescence intensity normalised to GAPDH for ERG, SMAD1, ENG, ID1, SMAD3, ALK5 and SMA ( n = 5). e Representative images of SMAD1 expression (white; arrows identify expression in the sinusoidal endothelium), VWF (green), SMA (red) and DAPI (blue) in liver tissue from Erg fl/fl and Erg cEC-het mice (Scale bar 50 μm). f Quantitative analysis of TGFβ2 protein expression was performed by ELISA in whole cell HUVEC lysates ( n = 7). g Representative image of TGFβ2 expression in HUVEC transfected with control or ERG siRNA by immunofluorescence; nuclei are identified by DRAQ V and cells are co-stained for ERG (green). Scale bar 20 µm. h Representative images of TGFβ2 expression (white; arrows) in large vessel within the liver from Erg fl/fl and Erg cEC-het mice (Scale bar 50 μm). Data were normalised to GAPDH and compared to control siRNA treated (*) by unpaired t -test. All graphical data are mean ± s.e.m., * P

    Journal: Nature Communications

    Article Title: Dynamic regulation of canonical TGFβ signalling by endothelial transcription factor ERG protects from liver fibrogenesis

    doi: 10.1038/s41467-017-01169-0

    Figure Lengend Snippet: Differentially expression of canonical TGFβ/BMP-SMAD genes in ERG-deficient HUVEC. a Microarray analysis of ERG-dependent genes in HUVEC was performed at 24 and 48 h after ERG depletion, as described ( n = 3 biological replicates) 24 ; fold change (log 2) of selected TGFβ/BMP associated genes represented as high (red) and low (blue) expression compared to the median (white). Gene expression data were validated in HUVEC transfected with Control (Con siRNA) or ERG siRNA by b quantitative PCR and c immunoblotting, showing reduction of ERG protein levels following siRNA, d quantification by fluorescence intensity normalised to GAPDH for ERG, SMAD1, ENG, ID1, SMAD3, ALK5 and SMA ( n = 5). e Representative images of SMAD1 expression (white; arrows identify expression in the sinusoidal endothelium), VWF (green), SMA (red) and DAPI (blue) in liver tissue from Erg fl/fl and Erg cEC-het mice (Scale bar 50 μm). f Quantitative analysis of TGFβ2 protein expression was performed by ELISA in whole cell HUVEC lysates ( n = 7). g Representative image of TGFβ2 expression in HUVEC transfected with control or ERG siRNA by immunofluorescence; nuclei are identified by DRAQ V and cells are co-stained for ERG (green). Scale bar 20 µm. h Representative images of TGFβ2 expression (white; arrows) in large vessel within the liver from Erg fl/fl and Erg cEC-het mice (Scale bar 50 μm). Data were normalised to GAPDH and compared to control siRNA treated (*) by unpaired t -test. All graphical data are mean ± s.e.m., * P

    Article Snippet: Twenty-four hours after co-transfection with the luciferase reporters and expression plasmids and 18 h after stimulation with human recombinant BMP9 (1 ng ml−1 , Peprotech) or TGFβ2 (10 ng ml−1 , Peprotech), HUVEC were lysed and reporter assays were performed in triplicate.

    Techniques: Expressing, Microarray, Transfection, Real-time Polymerase Chain Reaction, Fluorescence, Capillary Electrochromatography, Mouse Assay, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining

    ERG differentially regulates transcriptional activity of SMAD1 and SMAD3. ERG-dependent regulation of SMAD1 and SMAD3 activity were assessed by transactivation assays using either SMAD1 reporter pGL3-BRE (BRE reporter, a and c ) or SMAD3 reporter pBV-SBE4 (SBE reporter, b and d ). HUVEC were either co-transfected with Control siRNA (Con siRNA) or ERG siRNA for 24 h ( a and b ) or with pcDNA or ERG3 overexpression construct (ERG3) ( c and d ) prior to treatment with TGFβ2 (red) and BMP9 (blue) or PBS (dashed line) for 18 h (data from pooled HUVEC in triplicate experiments). The ratio of luciferase to renilla from each transfection was normalised to PBS-treated control or to groups co-transfected with ( a and b ) Con siRNA or ( c and d ) pcDNA ( n = 3). e Schematic of the inverse regulation of SMAD1 and SMAD3 by ERG. f Protein−protein interactions between ERG, SMAD1, SMAD2 and SMAD3 were assessed by Co-IP assay in whole cell lysate from HUVEC. SB-431542 (SB; 10 μM) treatment was performed for 1 h and TGFβ2 (10 ng ml −1 ) or PBS treatments were performed 30 min prior to lysis. Lysates were immunoprecipitated with mouse IgG or mouse α-ERG and then immuno-blotted for α-ERG, α-SMAD1, α-SMAD2, α-SMAD3 and α-pSMAD3 (Ser423/425). Images representative of four experiments. g ERG-SMAD3 interaction was assessed by Co-IP in untreated HSEC. h Cellular localisation of the interaction between ERG with pSMAD3 was investigated by Proximity Ligation Assay (PLA) following 30 min TGFβ2 treatment in HUVEC ( n = 2). i HUVEC or j HSEC were pre-treated with SB-431542 or DMSO prior to transfection with either control siRNA or ERG siRNA for 48 h and analysed by qPCR ( n = 3). Data were normalised to GAPDH and compared to control siRNA treated (*) or to ERG siRNA treated with DMSO (#) by unpaired t -test. All graphical data are mean ± s.e.m., * or # P

    Journal: Nature Communications

    Article Title: Dynamic regulation of canonical TGFβ signalling by endothelial transcription factor ERG protects from liver fibrogenesis

    doi: 10.1038/s41467-017-01169-0

    Figure Lengend Snippet: ERG differentially regulates transcriptional activity of SMAD1 and SMAD3. ERG-dependent regulation of SMAD1 and SMAD3 activity were assessed by transactivation assays using either SMAD1 reporter pGL3-BRE (BRE reporter, a and c ) or SMAD3 reporter pBV-SBE4 (SBE reporter, b and d ). HUVEC were either co-transfected with Control siRNA (Con siRNA) or ERG siRNA for 24 h ( a and b ) or with pcDNA or ERG3 overexpression construct (ERG3) ( c and d ) prior to treatment with TGFβ2 (red) and BMP9 (blue) or PBS (dashed line) for 18 h (data from pooled HUVEC in triplicate experiments). The ratio of luciferase to renilla from each transfection was normalised to PBS-treated control or to groups co-transfected with ( a and b ) Con siRNA or ( c and d ) pcDNA ( n = 3). e Schematic of the inverse regulation of SMAD1 and SMAD3 by ERG. f Protein−protein interactions between ERG, SMAD1, SMAD2 and SMAD3 were assessed by Co-IP assay in whole cell lysate from HUVEC. SB-431542 (SB; 10 μM) treatment was performed for 1 h and TGFβ2 (10 ng ml −1 ) or PBS treatments were performed 30 min prior to lysis. Lysates were immunoprecipitated with mouse IgG or mouse α-ERG and then immuno-blotted for α-ERG, α-SMAD1, α-SMAD2, α-SMAD3 and α-pSMAD3 (Ser423/425). Images representative of four experiments. g ERG-SMAD3 interaction was assessed by Co-IP in untreated HSEC. h Cellular localisation of the interaction between ERG with pSMAD3 was investigated by Proximity Ligation Assay (PLA) following 30 min TGFβ2 treatment in HUVEC ( n = 2). i HUVEC or j HSEC were pre-treated with SB-431542 or DMSO prior to transfection with either control siRNA or ERG siRNA for 48 h and analysed by qPCR ( n = 3). Data were normalised to GAPDH and compared to control siRNA treated (*) or to ERG siRNA treated with DMSO (#) by unpaired t -test. All graphical data are mean ± s.e.m., * or # P

    Article Snippet: Twenty-four hours after co-transfection with the luciferase reporters and expression plasmids and 18 h after stimulation with human recombinant BMP9 (1 ng ml−1 , Peprotech) or TGFβ2 (10 ng ml−1 , Peprotech), HUVEC were lysed and reporter assays were performed in triplicate.

    Techniques: Activity Assay, Transfection, Over Expression, Construct, Luciferase, Co-Immunoprecipitation Assay, Lysis, Immunoprecipitation, Proximity Ligation Assay, Real-time Polymerase Chain Reaction

    ERG is a key factor for endothelial specific inhibition of SMAD3-DNA binding. Putative ERG binding sites (grey bars) are located within the a TGβ2 and b CNN1 promoters upstream of the transcription start site (TSS) (arrow); ENCODE sequence conservation between 100 vertebrates across this region is shown. ENCODE ChIP-seq data profile for H3K4Me3, H3K27Ac, H3K9Ac and RNA polymerase II (RNA Pol2) in HUVEC indicate open chromatin and active transcription. Location of qPCR amplicon region R is indicated. ChIP-qPCR analysis of HUVEC was assessed following treatment with c and d TGFβ2 or PBS for 30 min, or e and f control or ERG siRNA. Chromatin was immunoprecipitated with c and e an α-ERG antibody, d and f α-SMAD3 antibody or control IgG. DNA was analysed by qPCR comparing specific primers against a negative control region for each target. Results are expressed as fold change compared to IgG ( n = 3 for all experiments). Basal enrichment was analysed compared to negative control region (#). TGFβ2 or ERG siRNA treatment were compared to PBS treated or control siRNA (*) mean ± s.e.m., * or # P

    Journal: Nature Communications

    Article Title: Dynamic regulation of canonical TGFβ signalling by endothelial transcription factor ERG protects from liver fibrogenesis

    doi: 10.1038/s41467-017-01169-0

    Figure Lengend Snippet: ERG is a key factor for endothelial specific inhibition of SMAD3-DNA binding. Putative ERG binding sites (grey bars) are located within the a TGβ2 and b CNN1 promoters upstream of the transcription start site (TSS) (arrow); ENCODE sequence conservation between 100 vertebrates across this region is shown. ENCODE ChIP-seq data profile for H3K4Me3, H3K27Ac, H3K9Ac and RNA polymerase II (RNA Pol2) in HUVEC indicate open chromatin and active transcription. Location of qPCR amplicon region R is indicated. ChIP-qPCR analysis of HUVEC was assessed following treatment with c and d TGFβ2 or PBS for 30 min, or e and f control or ERG siRNA. Chromatin was immunoprecipitated with c and e an α-ERG antibody, d and f α-SMAD3 antibody or control IgG. DNA was analysed by qPCR comparing specific primers against a negative control region for each target. Results are expressed as fold change compared to IgG ( n = 3 for all experiments). Basal enrichment was analysed compared to negative control region (#). TGFβ2 or ERG siRNA treatment were compared to PBS treated or control siRNA (*) mean ± s.e.m., * or # P

    Article Snippet: Twenty-four hours after co-transfection with the luciferase reporters and expression plasmids and 18 h after stimulation with human recombinant BMP9 (1 ng ml−1 , Peprotech) or TGFβ2 (10 ng ml−1 , Peprotech), HUVEC were lysed and reporter assays were performed in triplicate.

    Techniques: Inhibition, Binding Assay, Sequencing, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Amplification, Immunoprecipitation, Negative Control

    Dectin-1-activated DCs enhance Th9 cell differentiation in vitro . Naive CD4 + T cells from spleens of mice ( n =3–5) were cocultured with DCs matured with TNF-α/IL-1β (BMDC), Curlan (CurDC) or Scleroglucan (SclDC) in the presence of anti-CD3 with (Th9) or without (Th0) addition of Th9-polarizing cytokines TGF-β and IL-4 for 3 days. Culture supernatants and CD4 + T cells separated by the magnetic cell sorting (MACS) were collected for analysis. ( a ) Cells were stained with anti-CD4 and anti-IL-9 antibodies and subjected to flow cytometry analysis. Numbers in the dot plots represent the percentages of CD4 + IL-9 + T cells. Right, summarized results of four independent experiments obtained as at left. ( b ) ELISA assessed the IL-9 secretion in the cocultures. ( c , d ) qPCR analysis of Th9-, Th1-, Th2- and Th17-related cytokines ( c ) and transcription factors ( d ). Expression was normalized to Gapdh and set at 1 in BMDC-induced Th9 cells. Results shown are the mean±s.d. of 3–5 independent experiments. * P

    Journal: Nature Communications

    Article Title: Dectin-1-activated dendritic cells trigger potent antitumour immunity through the induction of Th9 cells

    doi: 10.1038/ncomms12368

    Figure Lengend Snippet: Dectin-1-activated DCs enhance Th9 cell differentiation in vitro . Naive CD4 + T cells from spleens of mice ( n =3–5) were cocultured with DCs matured with TNF-α/IL-1β (BMDC), Curlan (CurDC) or Scleroglucan (SclDC) in the presence of anti-CD3 with (Th9) or without (Th0) addition of Th9-polarizing cytokines TGF-β and IL-4 for 3 days. Culture supernatants and CD4 + T cells separated by the magnetic cell sorting (MACS) were collected for analysis. ( a ) Cells were stained with anti-CD4 and anti-IL-9 antibodies and subjected to flow cytometry analysis. Numbers in the dot plots represent the percentages of CD4 + IL-9 + T cells. Right, summarized results of four independent experiments obtained as at left. ( b ) ELISA assessed the IL-9 secretion in the cocultures. ( c , d ) qPCR analysis of Th9-, Th1-, Th2- and Th17-related cytokines ( c ) and transcription factors ( d ). Expression was normalized to Gapdh and set at 1 in BMDC-induced Th9 cells. Results shown are the mean±s.d. of 3–5 independent experiments. * P

    Article Snippet: Reagents Recombinant mouse GM-CSF, TNF-α, IL-1β, IL-4, IL-9 and human TGF-β were purchased from Peprotech.

    Techniques: Cell Differentiation, In Vitro, Mouse Assay, FACS, Magnetic Cell Separation, Staining, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Expressing