Tumor necrosis factor alpha tnf α

The human cytokines Interleukin 10 (IL-10), Transforming Growth Factor beta-1 (TGFβ1), Transforming Growth Factor beta-2 (TGFβ2), Transforming Growth Factor beta-3 (TGFβ3), and Tumor Necrosis Factor Alpha (TNFα) were purchased from Sigma-Aldrich, Interleukin 4 (IL-4) and Interferon Gamma (IFNy) from R&D Systems/Bio-Techne (R&D Systems/Bio-Techne, Wiesbaden-Nordenstadt, Germany), and Interleukin 6 (IL-6) and Vascular Endothelial Growth Factor165 (VEGF) from PeproTech (PeproTech, Winterhude, Germany). Porcine TGFβ3 was purchased from Biozol (Biozol, Eching, Germany), whereas porcine TGFβ1, TGFβ2, IL-4, IL-6, IL-10, IFNy and TNFα were from R&D System/Bio-Techne. Since there was no porcine VEGF available, the above mentioned human VEGF was also used for stimulation of pRMG.
To diminish the influence of cytokines present in FCS, both confluent pRMG and MIO-M1 cells were rinsed two times with prewarmed serum-free medium, followed by starvation for 1 h at 37°C and 5% CO2 with serum-deprived medium. Afterwards, cells were treated over night with IFNy, IL-4, IL-6, IL-10, TGFβ1, TGFβ2, TGFβ3, TNFα or VEGF165, respectively, in a randomized plate design at a concentration of 5 ng/ml in 2 ml medium without FCS. Untreated cells cultured in serum-free medium served as a control. For this study, cells were treated with each cytokine separately, but not with multiple cytokines in combination.

Human hepatoma HepG2 cells (ATCC, Manassas, VA, USA) were grown in Dulbecco’s modified Eagle’s medium (DMEM; Thermo Fisher, Pittsburgh, PA, USA) containing 10% fetal bovine serum (FBS; Atlanta Biologicals, Norcross, GA, USA) and 1% Penicillin–Streptomycin–Neomycin Antibiotic Mixture (Thermo Fisher, Pittsburgh, PA, USA) at 37 °C in 5% CO₂. HepG2 cells were treated with various concentrations (25–100 μM) of EPA (Nu-chek Prep, Inc., Elysian, MN, USA) conjugated with fatty acid free bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MO, USA) or BSA for 24 h and 48 h. Based on pilot studies, we used 50 μM for 24 h and it did not affect cell viability (not shown) but was effective on metabolic responses studied and thus used in additional experiments. In order to replicate inflammation induced by HF feeding in the mouse study, HepG2 cells were treated with 25 ng/mL tumor necrosis factor-alpha (TNF-α) (Sigma-Aldrich, St. Louis, MO, USA) for 4 h prior to being treated with TNF-α supplemented with 50 μM EPA for 6 h.

One day before transfection, TRBP knockout HeLa cells were inoculated at 2×10⁶ cells in a 9 cm dish. Cells were transfected with 3 μg of pGL2-TAR-luciferase or pGL2-ΔTAR-luciferase, along with pcDNA5-TRBP-WT, pcDNA5-TRBP-L326A/Y358A, or empty vector with Lipofectamine 2000 (Invitrogen). Four hours later, medium was exchanged into 10 ml of medium containing 10% FBS. Twenty-four hours after transfection, the cells were seeded at 2×10⁵ cells/well in a 12-well culture plate, and after 24 h, 20 ng of Tumor Necrosis Factor alpha (TNFα, Sigma-Aldrich) and 40 μg of cycloheximide (CHX, Wako) was added to the medium. At 6 and 10 h after TNFα/CHX treatment, the cells were photographed using Axiovert 200 microscope (ZEISS) and the cells were collected with Trypsin-EDTA. After washing the collected cells with PBS, 50 μl of RIPA Buffer was added and dissolved on ice for 20 min. After centrifugation at 7000×g for 10 min, the supernatant was mixed with 2×SDS-PAGE Sample Buffer and used for western blotting. In addition, total RNA was also recovered and the amount of mRNAs of GIT2 and IER3 genes were quantified using specific primers for each gene (Table S4).

Murine IL-1β, prepared in phosphate-buffered saline (PBS; HyClone, Logan, USA) containing 0.1% bovine serum albumin (BSA; Beyotime, Shanghai, China), was purchased from R&D Systems (Minneapolis, USA). Lipopolysaccharide (LPS) and tumor necrosis factor-alpha (TNF-α) were purchased from Sigma (St Louis, USA).

HeLa cells purchased from American Type Culture Collection (USA) were maintained in DMEM (Corning Mediatech, USA) supplemented with 10% fetal bovine serum (FBS; Corning Mediatech) and 1% penicillin/streptomycin. THP-1 cells were cultured in RPMI 1640 (Corning Mediatech) supplemented with 10% FBS, 2 mM glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 0.05 mM 2-mercaptoethanol, and 1% penicillin/streptomycin. Sodium arsenite (NaAsO2; AS), mitoTEMPO (MT), carbonyl cyanide chlorophenylhydrazone (CCCP), tumor necrosis factor-alpha (TNF-α) and cycloheximide (CHX) were purchased from Sigma-Aldrich (USA).
CVB3 and recombinant CVB3-GFP have been previously described [12 (link)]. Viruses were propagated in HeLa cells, and virus titration was conducted using plaque assays as previously described [13 (link)].

The following reagents and concentrations were used in experiments: 10 μg/mL Poly I:C (Sigma Aldrich, St. Louis, MO, USA), 100 μmol/L L-NG-Nitroarginine methyl ester (L-NAME, Sigma Aldrich, St. Louis, MO, USA), 10 μmol/L SB203580 (Cell Signaling Technology, Danvers, MA, USA). For a positive control of iNOS induction, 10 ng/mL tumor necrosis factor alpha (TNFα, Sigma Aldrich, St. Louis, MO, USA) was used. Agonists, inhibitors and vehicle controls were provided to cells in the presence of culture media.