The human bladder cancer cell line, UM-UC-3, was purchased from the American Type Culture Collection (Manassas, VA). Wild-type UM-UC-3 and its sublines were established in orthotopic xenograft models (primary bladder cancer, and lung, liver, and bone metastatic cells) cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS, Gibco®-life Technologies, Grand Island, NY, USA). Flufenamic acid and cisplatin were purchased from Sigma-Aldrich (St. Louis, MO). Human recombinant interleukin (IL)-6 and transforming growth factor-β1 (TGF-β1) were obtained from R&D Systems (Minneapolis, MN), and human recombinant IL-1β was purchased from PeproTech (Rocky Hill, NJ). Antibodies were purchased from the following suppliers: the antibody to aldo-keto reductase 1C1 (AKR1C1) was from Lifespan Biosciences (Seattle, WA); those for Snail, Slug, N-cadherin, phospho-FAK (Y397), phospho-Akt (S473), and phospho-Src (Y416) were from Cell Signaling Technology (Beverly, MA); those for fibronectin and Rac1 were from BD Transduction; and that for α-tubulin was purchased from Sigma-Aldrich.
Transforming growth factor β1 tgf β1
Manufactured by R&D Systems
Sourced in United States
Transforming growth factor-β1 (TGF-β1) is a multifunctional cytokine that plays a critical role in various cellular processes, including cell growth, differentiation, and immune regulation. It is a member of the transforming growth factor-beta family of proteins and is involved in the regulation of a wide range of biological activities.
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Lab products found in correlation
Human α thrombin, by Enzyme Research (3 mentions)
Tgf β1, by R&D Systems (2 mentions)
Optiprep, by Merck Group (2 mentions)
Phospho fak py397, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Recombinant human il 1β, by Thermo Fisher Scientific (1 mentions)
Phospho src y416, by Cell Signaling Technology (1 mentions)
Um uc 3, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
N cadherin, by Cell Signaling Technology (1 mentions)
Flufenamic acid, by Merck Group (1 mentions)
Cisplatin, by Merck Group (1 mentions)
Umuc3, by American Type Culture Collection (1 mentions)
Anti il 4, by R&D Systems (1 mentions)
Anti ifn γ, by R&D Systems (1 mentions)
Anti ifn γ, by BioXCell (1 mentions)
Anti cd3, by R&D Systems (1 mentions)
Anti cd28, by R&D Systems (1 mentions)
Anti il 4, by BioXCell (1 mentions)
Anti cd28, by BioXCell (1 mentions)
14 protocols using transforming growth factor β1 tgf β1
1
Establishment of Bladder Cancer Xenograft Models
2
Induction and Stability of Induced Regulatory T Cells
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Murine CD62L+ CD4+ T cells were magnetically sorted from freshly isolated splenocytes using a Miltenyi CD62L+ CD4+ T cell sorting kit according to the manufacturer’s instructions. Naive CD4+ T cells (3 × 105) were cultured on with RPMI 1640 medium supplemented with 10% fetal bovine serum, penicillin/streptomycin, and β-mercaptoethanol in a 48-well plate precoated with goat anti-hamster cross-linking antibody. To obtain iTreg cells in vitro, the medium was supplemented with anti-CD3 (0.25 μg/ml, 145-2C11; BioLegend, San Diego, CA), anti-CD28 (0.5 μg/ml, 37.51; BioXcell, Lebanon, NH), IL-2 (20 ng/ml; R&D Systems), transforming growth factor–β1 (TGF-β1; 1.0 ng/ml; R&D Systems), anti–IFN-γ (10 μg/ml; BioXcell), and anti–IL-4 (10 μg/ml; BioXcell). To evaluate iTreg stability, day 3–differentiated iTreg cells were cultured 24 hours with TH17-differentiating conditions: anti-CD3 (0.25 μg/ml), anti-CD28 (0.5 μg/ml), IL-6 (3 ng/ml; R&D Systems), TGF-β1 (0.3 ng/ml), anti–IFN-γ (10 μg/ml), and anti–IL-4 (10 μg/ml).
3
Characterization of A549 cells and signaling pathways
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Commercially-available A549 cells were characterised and sourced from ATCC (LGC Standards, UK). Human α-thrombin was purchased from Enzyme Research Laboratories, UK. Transforming growth factor β-1 (TGFβ-1) was purchased from R&D Biosystems, UK. PAR-1 agonist peptide TFLLR-NH2 was purchased from Bachem AG, Switzerland. Hirudin was purchased from Sigma, UK. MEK inhibitor, UO126, and ALK5 inhibitor, SB431542 were purchased from Calbiochem, UK. PAR-1 ATAP2 detection antibody was purchased from Santa Cruz Biotechnology, USA. Total Smad2 and Smad3, and phosphorylated Smad2 and Smad3, ERK and phosphorylation ERK antibodies were purchased from Cell Signalling Technologies, USA. PAR-1 inhibitor, RWJ58259, was synthesised in-house by the Department of Chemistry, UCL. Mithramycin A was purchased from VWR International, UK and WP631 was purchased from Insight Biotechnology, UK.
4
Regulation of Fibrosis Pathways
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Transforming growth factor-β1 (TGF-β1) and platelet derived growth factor BB (PDGF-BB) were obtained from R&D Systems (Minneapolis, MN). The following antibodies were purchased: phospho-FAK [pY397] (Biosource, Camarillo, CA), procollagen alpha 1 type 1 (1A1), Fyn, Cyclin D1 (Santa Cruz Biotechnology, Santa Cruz, CA), FAK (N-terminal domain) (Santa Cruz, CA), Fyn and phospho-SrcY418 (Cell Signaling Technology, Boston, MA), phosphor-ERK and ERK, phosphor-p38 and p38, Cleaved caspase-3, cleaved Poly-(ADP-ribose) polymerase (PARP), (EMD Millipore, Billerica, MA), and glyceraldehyde 3-phosphate dehydrogenase (G3PDH) (Research Diagnostics, Flanders, NJ). Anti-α-SMA antibody, Cy3-conjugated Anti-α-SMA antibody, carbon tetrachloride (CCl4), corn oil, and OptiPrep were purchased from Sigma-Aldrich (St. Louis, MO). FAK inhibitor (PF-562271) was purchased from Selleckchem and Fisher Scientific (Waltham, MA). Kits for active Rac and Rho were purchased from EMD Millipore (Billerica, MA). The generation and production of adenoviral vectors containing FAK-related non-kinase (ad-FRNK) or control green fluorescent protein (GFP) were described previously26 (link). All other chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO) and Fisher Scientific (Waltham, MA).
5
Investigating TGF-β1 and Kinase Inhibitor Effects on HK-2 Cells
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HK-2 cells (human renal proximal tubular epithelial cells)54 (link) (ATCC® CRL-2190™) were maintained in DMEM/F12 media (Gibco™) supplemented with 2% FBS, Hepes buffer, insulin, transferrin, sodium selenite, glucose, dexamethasone, EGF, penicillin, and streptomycin (Sigma Aldrich). Fresh growth medium was changed every 2–3 days. Before treatments, cells were growth arrested in serum-free medium and incubated separately with serum-free medium (control), transforming growth factor-β1 (TGF-β1: 1, 2, 4 ng/ml; R&D Systems, Minneapolis, MN) and/or U0126 (10 µM/L; #662005, Calbiochem), SB203580 (20 µM/L; #559389, Calbiochem), LY294002 (10 µM/L; #440202, Calbiochem) for different periods of time. Cells were maintained according to the described protocol, unless otherwise indicated.
6
Oridonin Analogue CYD0692 Inhibits Fibrosis
7
Colon Cancer Cell Line Culture and EMT Induction
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Colon cancer cell lines (HCT116, SW480, HCT-8, and LS174T) and the human normal colonic epithelial cell line CCD 841 CoN were purchased from the American Tissue Culture Collection (ATCC; Mannassas, VA, USA) and cultured in RPMI-1640 medium (Gibco, Rockville, MD, USA) supplemented with 10% fetal bovine serum (FBS) at 37°C with 5% CO2. Transforming growth factor-β1 (TGF-β1) (4 ng/ml; R&D Systems, Minneapolis, MN, USA), an EMT inducer, was used to treat transfected colon cancer cells for 24 h. LiCl (20 mM; Sigma-Aldrich, St. Louis, MO, USA), a Wnt/β-catenin pathway activator, was used to treat transfected colon cancer cells for 24 h.
8
MiR-210-3p Modulation of TGF-β1 Response
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After 24 h of cell seeding, cells were transfected for 24 h in Opti‐MEM Reduced Serum Medium (Thermo Fisher Scientific) using a miR‐210‐3p mimic (Thermo Fisher Scientific), negative control (Thermo Fisher Scientific), and Lipofectamine 2000 (Thermo Fisher Scientific) according to the instructions provided by the manufacturer. The mimic complexes were transfected into cells at a final concentration of 50 nM. The transfection medium was replaced with fresh medium 24 h later, and cells were incubated for another 48 h. Transforming growth factor‐β1 (TGF‐β1) was obtained from R&D Systems, Inc. Cells were exposed to 5 ng/ml TGF‐β1 for 48 h.
9
Transforming Growth Factor-β1 Signaling
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Transforming growth factor-β1 (TGF-β1) was obtained from R&D Systems (Minneapolis, MN). The following antibodies were purchased: phospho-FAK [pY397] (Biosource, Camarillo, CA), procollagen alpha 1 type 1 (1A1) and fibronectin (Santa Cruz Biotechnology, Santa Cruz, CA), FAK (Cell Signaling, MA), α-SMA (American Research Products, Belmont, MA), and glyceraldehyde 3-phosphate dehydrogenase (G3PDH) (
10
Chondrogenic Differentiation of BMSCs
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At passages 3–5, 2.5 × 105 adult or infant BMSCs were incubated in an induction medium contained the serum-free and high-glucose (4.5 g/l) Dulbecco’s modified Eagle’s medium (HG-DMEM) (Gibco, Grand Island, NY, USA) with 10-7 M dexamethasone (Sigma-Aldrich), 50 μg/ml ascobate-2-phosphate (Sigma-Aldrich), 50 μg/ml L -proline, 10 ng/ml transforming growth factor-β1 (TGF-β1) (R&D Systems, Minneapolis, MN, USA) and Insulin-Transferrin-Selenium (ITS) plus Premix (BD Biosciences, San Jose, CA, USA). After centrifugation at 1500 rpm for 5 min to form a pellet, the sample was transferred into a culture incubator for cultivation and cultured in 5% CO2 at 37°C. The medium changed every 3 days, and the chondrogenic pellets were observed and harvested at days 7, 14, and 21 for RT-qPCR detection during the induction21
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