Compound c

Murine skeletal muscle C2C12 cells (American Type Culture Collection, Manassas, VA, USA) were cultured in Dulbecco’s Modified Eagle’s Medium containing 10% fetal bovine serum and incubated at 37 °C in a humidified CO₂ incubator. Cells were cultured until they reached 90% confluence, and differentiation was initiated by exchanging the medium with Dulbecco’s Modified Eagle’s Medium containing 2% horse serum (Gibco, Grand Island, NE, USA; differentiation medium). G-Rf (10, 20, and 40 μM) dissolved in differentiation medium was added to the cells for 48 or 24 h on day 2 or 5 of differentiation, respectively (Figure 2A). Cells were treated with specific inhibitors of AMPK and p38 (compound C and SB203580, respectively; all 10 μM; Cayman Chemicals) for 24 h on day 5 of differentiation. DMSO-treated cells were used as a control and were compared with the experimental groups.

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 (ERK^1/2 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). N_ω-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.

LPS (from Escherichia coli, 055:B5), D-Gal and pifithrin-α were the products of Sigma (St. Louis, MO, USA). Compound C and A-769662 were the products of Cayman Chemical (Ann Arbor, MI, USA). SP600125 was the product of Enzo life sciences (New York, NY, USA). The kits for the determination of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were produced by Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The enzyme-linked immunosorbent assay (ELISA) kits for determination of mouse TNF-α and interleukin 6 (IL-6) were produced by NeoBioscience Technology Company (Shenzhen, China). The total protein extract kit and the colorimetric assay kits for caspase-3, -8, -9 were produced by Beyotime Institute of Biotechnology (Jiangsu, China). The In Situ Cell Death Detection Kit was produced by Roche (Indianapolis, IN, USA). The rabbit anti-mouse AMPK, phospho-AMPKα (Thr¹⁷²), c-Jun N-terminal kinase (JNK), phospho-JNK (Thr¹⁸³/ Thr¹⁸⁵), cleaved caspase-3 and β-actin antibodies were the products of Cell Signaling Technology (Danvers, MA, USA). The BCA protein assay kit, the horseradish peroxidase-conjugated goat anti-rabbit antibody and the enhanced chemiluminescence (ECL) reagents were the products of Pierce Biotechnology (Rockford, IL, USA).

Compound C (Cayman Chemicals) was suspended in dimethyl sulfoxide (DMSO) at a stock concentration of 10 μM and added to J774A.1 macrophages at the indicated concentrations 1 h prior to bacterial inoculation. The Compound C dosage was determined based on data from previous studies (50 (link)). Compound C remained on macrophages throughout infection. The cytotoxicity of Compound C in J774A.1 macrophages was determined using a Vybrant 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) cell proliferation assay kit (Thermo Fisher) according to the manufacturer’s protocol. Atglistatin (30 μM) (Cayman Chemicals), triacsin C (10 μM) (Enzo Life Sciences), and T863 (10 μM) (Cayman Chemicals) were suspended in DMSO (12 (link), 30 (link)). For lipid inhibitor studies, we pretreated cells overnight with compounds and continued treatment throughout infection.