Dmem high glucose

Male Sprague-Dawley (SD) rats (n=10; weight, 50±1.4 g; 4-week-old) were purchased from Shanghai SIPPR-Bk Lab Animal Co., Ltd. (Shanghai, China) with the certificate number 2008001682174. The animal license was provided by Shanghai Laboratory Animal Center (Shanghai, China) with the license number SCXK 2013–0016. Rats were housed at 25°C with a 12:12 h light/dark for a week, and given healthy atmosphere air, food and water ad libitum. Rats were sacrificed by cervical dislocation and cartilage tissue was harvested from knee joints, which was immersed in PBS, cut into slices (2–4 mm thick) and placed in 5 ml centrifuge tubes. The use and treatment of SD rats was approved by the Ethics Committee of the School of Medicine, Shandong University (Jinan, China). Chondrocytes were obtained by enzyme digestion using collagenase II and were then maintained in high glucose DMEM with 15% FBS (v/v; both GE Healthcare Life Sciences) at 37°C under 5% CO₂ until a confluence of 50–60% was reached. Immunohistochemical staining (IHC) was subsequently performed for identification

The experiment was carried out as previously described¹⁹ with modifications. C2C12 cells were cultured and switched to differentiation medium (DM) for 4 days. The DM for C2C12 cells was high‐glucose DMEM with 2% horse serum (GE Healthcare, Chicago, Illinois, USA). After 4 days in differentiation, cells were then treated with DM (as a control), 50% shIFIT2 conditioned medium (CM) in DM with or without neutralizing IL6 Antibody (#MAB2061; R&D systems, Minneapolis, MN, USA) for 3 days. Control and experimental cells were given fresh medium every 24 h. Myotubes were washed with PBS and fixed with ice‐cold 100% methanol. Fixed cells were permeabilized with 0.3% Triton X‐100, blocked in 2% BSA‐PBS for 1 h at room temperature and immuostained with Alexa Fluor 488‐conjugated Myosin 4 Monoclonal Antibody overnight at 4°C (#53‐6503‐82; 1:500; Thermo Fisher Scientific). Myotubes were photographed by a DS‐Qi2 camera with NIS‐Elements Software (Nikon, Tokyo, Japan). Average myotube diameter per well was measured in a total of 50 myotubes from at least 5 random fields using ImageJ software (NIH). Three independent wells were used to calculate mean values for control and treated myotubes.

Recombinant Human tumor necrosis factor (TNF)-α and IL-1β were obtained from PeproTech China. Fetal bovine serum and high-glucose DMEM were purchased from HyClone; GE Healthcare Life Sciences. Trypsin-EDTA solution and the Trypan Blue Staining Cell Viability Assay kit were purchased from Beyotime Institute of Biotechnology. The RNA PCR kit (EX TAQ R-PCR Version 2.1) was obtained from Takara Biotechnology Co., Ltd., and primers and probes were purchased from Sangon Biotech Co., Ltd. TRIzol^® reagent was purchased from Invitrogen. Asarinin was purchased from Chengdu Must Bio-Technology Co., Ltd., and ELISA kits for TNF-α (cat. no. 555212), IL-6 (cat. no. 555220) and IFN-γ (cat. no. 555142) from BD Biosciences. The ELISA kit for IL-17A (cat. no. D1700) was obtained from R&D Systems, Inc. Primary antibodies for TNF-α (cat. no. TA808184), IL-17A (cat. no. TA337063), IL-6 (cat. no. TA500067) and IFN-γ (cat. no. TA353236) were from OriGene Technologies, Inc. Peptidoglycan (PGN) and lipopolysaccharide (LPS) were purchased from Sigma-Aldrich.

Liver cancer HepG2 cells, purchased from the American Type Culture Collection, were cultured in high-glucose DMEM (GE Healthcare Life Sciences) with 10% fetal bovine serum (Clark Bioscience) and 1% penicillin/streptomycin at 37°C in an atmosphere containing 5% CO₂. The cells were dissociated with 0.25% trypsin (w/v) and 0.52 mM EDTA [M&C Gene Technology (Bejing) Ltd.] and routinely sub-cultured at 80% confluency.
Cell cultures were treated with various concentrations of apigenin in DMSO as the carrier solvent. Palmitic acid binds to fatty-acid-free BSA (Beijing Solarbio Science & Technology Co., Ltd.). In brief, palmitic acid was dissolved in 1X PBS and a 250 mM stock solution was obtained following various cycles of incubation in a water bath at 70°C and vortexing. The stock solution was then added to serum-free DMEM containing 5% fatty-acid-free BSA to obtain a 250 µM palmitic acid solution, and the resulting diluted solution was used for the cell treatments (16 (link),17 (link)). AMPK inhibitor compound C was diluted with DMSO to a final concentration of 10 µM and was used to verify the signaling pathway.