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3 protocols using recombinant mouse tnf α

1

Investigating AVAs and TNF-α in C2C12 Cells

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C2C12 cells were obtained from the American Type Culture Collection (CRL-1772: Manassas, VA, USA) and cultured in Dulbecco's modified minimum essential medium (DMEM) supplemented with 20% fetal bovine serum (Gibco, Carlsbad, CA, USA) and penicillin:streptomycin solution (50U/mL and 50 µg/mL, respectively; Gibco) at 37°C in air with a humidified atmosphere of 5% CO2. An AVA stock solution was made in dimethyl sulfoxide (DMSO). The final concentration of DMSO in the cultured medium was 0.05%. A total of 30 μmol synthetic AVAs (AVA-A, -B, and -C) provided by Dr. Mitchell Wise (USDA Cereal Research Laboratory, Madison, WI, USA) were treated with fresh medium, when the cells reached 70% confluence. An equivalent amount of DMSO was added to the control cells. After the AVA was treated for 24 h, recombinant mouse TNF-α (Roche, Basel, Switzerland) was added to a final concentration of 10ng/mL for various incubation times depending on the experimental protocols.
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2

Calcium Signaling Modulators Protocol

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TRPV4 specific agonist GSK1016790A (GSK101, #G0798) and inhibitor GSK2193874 (GSK874, #5106) were obtained from Sigma-Aldrich (St. Louis, MO) and Tocris Bioscience (Bristol, UK), respectively. Compound C (AMPK inhibitor, #11967), STO-609 (calmodulin-dependent kinase kinase (CaMKK) inhibitor, #15325), Wortmannin (PI3K inhibitor, #10010591), LY294002 (Akt inhibitor, #70920), U0126 (ERK1/2 inhibitor, #70970), AICAR (#10010241), Forskolin (#11018), and Phorbol 12-myristate 13-acetate (PMA, #10008014) were obtained from Cayman Chemical (Ann Arbor, MI). Ruthenium Red (Ca2+ fluxes blocker, #557450), and H-89 (PKA inhibitor, #371963) were obtained from Calbiochem (San Diego, CA). Calcium chelator BAPTA/AM (#B-1205) was from Invitrogen (Grand Island, NY). Recombinant mouse TNF-α was from Roche (#11271156001, Indianapolis, IN). Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME, #N5751), Sodium nitroprusside (SNP, #PHR1423), Acetylcholine chloride (#A2661), (R)-(−)-Phenylephrine hydrochloride (PE, #P8155) and other chemicals were obtained from Sigma-Aldrich.
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3

Rat Myogenic Cells Differentiation

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L6 rat myogenic cells were seeded at the density of 25,000/cm2 and cultured in growth medium (GM) consisting of: Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin. The cells were incubated at 37 °C in a humidified atmosphere with 5% carbon dioxide and 95% air. Twenty-four hours after plating, cultures were washed with Phosphate Buffer Saline (PBS) and shifted to low-serum medium consisting of DMEM supplemented with 1% fatty acid-free bovine serum albumin (BSA, #A9418 Sigma, St. Louis, MO, USA) [42 (link)] and treated with 200 μM Taurine (#T8691 Sigma, St. Louis, MO, USA) and/or 15 ng/mL recombinant mouse TNF-α (#11271156001; Roche, Indianapolis, IN, USA) at different times as described below.
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