The presence of Activin A and TGFβ in the supernatants of cultured cells was determined by ELISA carried out following standard procedures, according to the manufacturers’ instructions (TGFβ – Promega, Southampton, UK; Activin A – R&D Systems, Abingdon, UK). Cells were cultured in serum-free medium supplemented with 0.5% BSA for 16 h prior to collection of conditioned medium.
Tgf β
Manufactured by Promega
Sourced in United States, United Kingdom
TGF-β is a multipurpose lab equipment designed for the analysis and detection of transforming growth factor-beta proteins. It provides researchers with the tools to quantify and study this important signaling molecule, which plays a crucial role in various biological processes, such as cell growth, differentiation, and immune function.
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Lab products found in correlation
Glutamine, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Xmd8 92, by Selleck Chemicals (1 mentions)
Tgf β1, by Thermo Fisher Scientific (1 mentions)
Celltiter 96 aqueous one solution cell proliferation assay, by Promega (1 mentions)
Neutrophil elastase, by R&D Systems (1 mentions)
Enzyme linked immunosorbent assay kit, by MyBioSource (1 mentions)
Cyclin d1, by Novus Biologicals (1 mentions)
Oxiselecttm sta 347 in vivo ros rns assay kit, by Cell Biolabs (1 mentions)
Corticosterone, by Assaypro (1 mentions)
Microplate reader, by Molecular Devices (1 mentions)
Tnf α, by R&D Systems (1 mentions)
Ifn γ, by R&D Systems (1 mentions)
Interleukin 4 (il 4), by R&D Systems (1 mentions)
β endorphin, by Phoenix Pharmaceuticals (1 mentions)
α msh, by Phoenix Pharmaceuticals (1 mentions)
Activin a, by Promega (1 mentions)
Activin a, by R&D Systems (1 mentions)
Tgf β2, by R&D Systems (1 mentions)
Transfectin lipid reagent, by Bio-Rad (1 mentions)
9 protocols using tgf β
1
Quantification of Activin A and TGFβ
2
Cytokine Profiling of NK Cells and PBMC After E. multilocularis-VF Stimulation
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Before and after stimulation of NK cells and PBMC with E. multilocularis-VF, supernatants were collected for ELISA testing cytokines. For NK cells, the secretion of two cytokines was measured: interferon γ (IFN-γ) (Diaclone, Besançon, France) and transforming growth factor β (TGF-β) (Promega, Charbonnières les Bains, France). For PBMC, the secretion of four cytokines was measured: IFN-γ, IL-10 and IL-17 (Diaclone) and TGF-β (Promega). Each sample was measured in duplicate, following the manufacturer's instructions. The TGF-β assay used allowed a specific detection of TGF-β 1 , with less than 3% cross-reactivity with other related growth factors (TGF-β 2 and TGF-β 3 ).
3
Immortalized Murine Liver Cell Lines
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Resident liver stem cells (RLSCs) and HepE14 hepatocytes are immortalized, and non-tumorigenic cell lines derived from murine liver explants at 14th days of development [60 (link)–62 (link)]. RLSC and HepE14 were grown as previously described [39 (link)].
The human liver carcinoma cell lines HuH7 and HepG2 were grown at 37 °C, in a humidified atmosphere with 5% CO2 on plastic (Corning) in Dulbecco’s modified Eagle’s medium (DMEM; Gibco-Life Technologies), supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine (Gibco-Life Technologies) and antibiotics (Gibco-Life Technologies).
For the experiments in 3D cell culture, HuH7 cells were grown in a low attachment substrate (0.6% agar) until the formation of suspended aggregates/spheroids (24 h).
Where indicated, cells were treated with 10 µM ERK5 inhibitor XMD8-92 (Selleckchem, Selleck Chemicals GmbH), 10 µM or 20 µM MEK5 inhibitor BIX02189 (Selleckchem, Selleck Chemicals GmbH), 10 µM of YAP-TEAD inhibitor Verteporfin or 4 ng/ml of TGFβ1 (PeproTech Inc., Rocky Hill, NJ, USA) for the indicated time. As previously reported, hepatocytes utilized in this study undergo EMT following TGFβ treatment [63 (link)–65 (link)].
The number of viable cells upon 16 h of treatment with MEK5/ERK5 inhibitors has been analyzed by CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega), following the manufacturer’s protocol.
The human liver carcinoma cell lines HuH7 and HepG2 were grown at 37 °C, in a humidified atmosphere with 5% CO2 on plastic (Corning) in Dulbecco’s modified Eagle’s medium (DMEM; Gibco-Life Technologies), supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine (Gibco-Life Technologies) and antibiotics (Gibco-Life Technologies).
For the experiments in 3D cell culture, HuH7 cells were grown in a low attachment substrate (0.6% agar) until the formation of suspended aggregates/spheroids (24 h).
Where indicated, cells were treated with 10 µM ERK5 inhibitor XMD8-92 (Selleckchem, Selleck Chemicals GmbH), 10 µM or 20 µM MEK5 inhibitor BIX02189 (Selleckchem, Selleck Chemicals GmbH), 10 µM of YAP-TEAD inhibitor Verteporfin or 4 ng/ml of TGFβ1 (PeproTech Inc., Rocky Hill, NJ, USA) for the indicated time. As previously reported, hepatocytes utilized in this study undergo EMT following TGFβ treatment [63 (link)–65 (link)].
The number of viable cells upon 16 h of treatment with MEK5/ERK5 inhibitors has been analyzed by CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega), following the manufacturer’s protocol.
4
Biomarker Profiling for Kidney Injury
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The biomarkers were quantified by ELISA at the end of each time period (7, 14 and 21 days) using the 24-hour urine.
KIM-1 (RKM 100, R&D Systems, Minneapolis, USA) was used to identify proximal tubular and acute kidney injury. NGAL (KIT 046, BioPorto, Denmark) was used as a tubular and acute kidney injury biomarker. Fibronectin (KT-415, Kamiya, WA, USA) and TGF-β (G7590, Promega, WI, USA) were used as fibrosis biomarkers. Osteopontin (ADI-900-090A, Enzo, PA, USA), TNF-α (CKR041, Cell Sciences, MA, USA) and IL-6 (DY506, R&D Systems, Minneapolis, USA) were used as markers of macrophage infiltration and inflammation, and podocin (E90938Ra, Uscn Life Science Inc., P.R. China) served as a glomerular damage biomarker.
KIM-1 (RKM 100, R&D Systems, Minneapolis, USA) was used to identify proximal tubular and acute kidney injury. NGAL (KIT 046, BioPorto, Denmark) was used as a tubular and acute kidney injury biomarker. Fibronectin (KT-415, Kamiya, WA, USA) and TGF-β (G7590, Promega, WI, USA) were used as fibrosis biomarkers. Osteopontin (ADI-900-090A, Enzo, PA, USA), TNF-α (CKR041, Cell Sciences, MA, USA) and IL-6 (DY506, R&D Systems, Minneapolis, USA) were used as markers of macrophage infiltration and inflammation, and podocin (E90938Ra, Uscn Life Science Inc., P.R. China) served as a glomerular damage biomarker.
5
Biomarker Profiling of Skin and Plasma in Experimental Model
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The blood and dorsal skin samples were collected on the last experimental day. The plasma was separated from the blood samples via centrifugation at 3,000× g and 4 °C for 10 min, and the supernatant was used for further analysis. The levels of IL-10, TGF-β, IL-6, and IL-23 were determined using commercial enzyme-linked immunosorbent assay kits (IL-10, IL-6, and IL-23; MyBiosource, San Diego, CA, USA; TGF-β; Promega, Madison, MI, USA) as per the manufacturer’s instructions. We first rinsed 10 mg of dorsal skin in phosphate-buffered saline to remove excess blood before homogenization. The samples were then homogenized at 1500 × g and 4 °C for 15 min, and the supernatant was collected for assaying. The levels of Ki67, cyclin D1, and neutrophil elastase in the dorsal skin were determined using commercial enzyme-linked immunosorbent assay (ELISA) kits (Ki67; Wuhan Fine Biotech, Hubei, China; cyclin D1; Novus, Centennial, CA, USA; neutrophil elastase; R&D Systems, Inc., Minneapolis, MN, USA) according to the manufacturers’ instructions. The ROS levels in the dorsal skin were determined with an OxiSelectTM STA-347 in vivo ROS/RNS assay kit (Cell Biolabs, Inc., San Diego, CA, USA) in accordance with the manufacturer’s instructions. Optical density was measured using a microplate reader (Mdecular Devices, Sunnyvale, CA, USA).
6
Plasma Biomarkers in Mice Post-Exercise
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Blood samples were obtained from the hearts of the mice two weeks after the treadmill treatment. The plasma levels of ACTH, β-endorphin, α-melanocyte stimulating hormone (α-MSH), corticosterone, IgE, transforming growth factor (TGF)-β, histamine, tumor necrosis factor (TNF)-α, interleukin (IL)-4, IL-10, interferon (IFN)-γ, and IL-13 were determined using commercial ELISA kits (ACTH, β-endorphin and α-MSH: Phoenix Pharmaceuticals Inc., Burlingame, CA, USA; corticosterone: Assaypro, St. Charles, MO, USA; IgE: Yamasa Shoyu Co., Ltd., Chiba, Japan; TGF-β: Promega, Madison, WI, USA; histamine: Bertin Pharma, Montigny le Bretonneux, France; TNF-α, IL-4, IL-10, IFN-γ, and IL-13: R&D Systems, Minneapolis, MN, USA) according to the manufacturers’ instructions. The optical density was measured with a microplate reader (Molecular Devices, Sunny Vale, GA, USA).
7
TGF-β Signaling Pathway Activation Assay
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Cells were seeded at a density of 2 X 104 cells/well in 12-well tissue culture plates. The following day, the cells were transiently transfected using Transfectin lipid reagent following the manufacturer’s protocol (Bio-Rad #170-3351, Hercules, CA, USA). Cells were transfected with 1.5 μg 3TP-Lux
[22 (link)] or CAGA(9)-Luc
[23 (link)]. pRL-CMV-renilla (Promega #E2261, Madison, WI, USA) was co-transfected and used as an internal control to correct for transfection efficiency. Eighteen hours after transfection, cells were treated with 1 ng/ml TGF-β1 or TGFβ-2 (R&D Systems, #102-B1 and #102-B2, respectively). Twenty-four hours after TGF-β treatment, cells were harvested and assayed for promoter specific luciferase activity using a Dual-Luciferase Reporter Assay System (Promega #E1910) according to the manufacturer’s protocol. Luciferase activity was measured using a BD/Pharmigen Monolight 3010 luminometer.
[22 (link)] or CAGA(9)-Luc
[23 (link)]. pRL-CMV-renilla (Promega #E2261, Madison, WI, USA) was co-transfected and used as an internal control to correct for transfection efficiency. Eighteen hours after transfection, cells were treated with 1 ng/ml TGF-β1 or TGFβ-2 (R&D Systems, #102-B1 and #102-B2, respectively). Twenty-four hours after TGF-β treatment, cells were harvested and assayed for promoter specific luciferase activity using a Dual-Luciferase Reporter Assay System (Promega #E1910) according to the manufacturer’s protocol. Luciferase activity was measured using a BD/Pharmigen Monolight 3010 luminometer.
8
Modulating HUVEC and HeLa Cell Behavior
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Primary human umbilical vein ECs (HUVECs) were cultured in an EC medium (ScienCell) supplemented with 5% FBS and EC growth supplements. Cells were used in experiments between passages 2 and 5. HeLa cells were maintained in DMEM supplemented with 10% FCS. The γ‐secretase inhibitor (Alexis Biochemicals), VEGF, and TGF‐β (Promega) were used at concentrations of 25, 30, and 2.5 ng/mL, respectively. Inhibitors of Akt (LY294002) and ERK (ERK inhibitor) were purchased from Cayman Chemical Company and used at the concentrations of 10 and 20 μmol/L, respectively.
HUVECs were transfected with miR‐342‐5p mimics or control or miR‐342‐5p antisense oligonucleotides (ASO) or control (RiboBio) at a concentration of 50 or 100 nmol/L by using Lipofectamin LTX reagent (Invitrogen), according to the manufacturer's instructions. The coding region of murine endoglin cDNA was cloned by polymerase chain reaction (PCR) and inserted into pcDNA3 to generate pcDNA3–mouse endoglin for transfection with Lipofectamin 2000. In some experiments, cells were transduced with lentivirus particles expressing miR‐342‐5p or control, which were purchased from Shanghai Genechem Co and handled according to the provider's protocols.
HUVECs were transfected with miR‐342‐5p mimics or control or miR‐342‐5p antisense oligonucleotides (ASO) or control (RiboBio) at a concentration of 50 or 100 nmol/L by using Lipofectamin LTX reagent (Invitrogen), according to the manufacturer's instructions. The coding region of murine endoglin cDNA was cloned by polymerase chain reaction (PCR) and inserted into pcDNA3 to generate pcDNA3–mouse endoglin for transfection with Lipofectamin 2000. In some experiments, cells were transduced with lentivirus particles expressing miR‐342‐5p or control, which were purchased from Shanghai Genechem Co and handled according to the provider's protocols.
9
Quantifying Inflammatory Biomarkers by ELISA
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Levels of 25(OH)D3 (Cayman Chemical, Michigan, USA), TGFβ (Boster, Pleasanton, CA, USA), IL-35 (Elabscience, Houston, USA), IL-17A (Diaclone, Besancon, FR), and MMP9 (BioVendor, Brno, CZ) were assayed by the ELISA technique using a manufacturer kit. The limits of detection were 25(OH)D3 and MMP9, <0.5 ng/mL; TGFβ, IL-35, and IL-17A ≤ 1 pg/mL. For all assays, absorbance was assessed using a Glomax multidetection reader spectrophotometer (Promega, Milan, Italy).
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