Sto 609

The C2C12 cell line used in this study was purchased from American Type Culture Collection (ATCC). C2C12 cells were cultured in DMEM/HIGH GLUCOSE (Hyclone) with 10% foetal bovine serum (Gibco). C2C12 cells were seeded in 24‐well plates (4 × 10⁵ /cm²). After 24 h, we treated the C2C12 cells with TNF‐α (MedChemExpress, Monmouth Junction, USA) at the concentration of 1 ng/ml for 7 days to induce C2C12 cell muscle atrophy. Dissolve 3.2 mg DHM into 10 ml DMSO to prepare a 1 mM DHM solution. In C2C12 cell experiments, the DHM solution was mixed into the cell culture medium at a ratio of 1/1,000. We treated C2C12 cells with TNF‐α and DHM (1 μM, purity ≥ 98%, Sigma Chemical Inc.) for 7 days to demonstrate DHM resisted inflammation‐induced muscle atrophy. We treated C2C12 cells with TNF‐α, DHM and Compound C (5 μM, MedChemExpress) for 7 days to demonstrate DHM resisted inflammation‐induced muscle atrophy through AMPK. We treated C2C12 cells with TNF‐α, DHM and STO‐609 (10 ng/ml, MedChemExpress) for 7 days to demonstrate DHM‐resisted inflammation‐induced muscle atrophy through CaMKK. We treated C2C12 cells with TNF‐α, DHM and ryanodine (100 nM, MedChemExpress) for 7 days to demonstrate DHM resisted inflammation‐induced muscle atrophy through the ryanodine receptor.

The 3T3-L1 cell line used in this study was purchased from American Type Culture Collection (ATCC, VA, USA). 3T3-L1 was cultured in DMEM/High Glucose (Hyclone, PA, USA) with 10% Fetal Bovine Serum (Gibco, NY, USA). 3T3-L1 cells were seeded in 24-well plates (4 × 10⁵/cm²). After 24 h, we treated the 3T3-L1 with TNFα (MedChemExpress, Monmouth Junction, USA) at the concentration of 1 ng/mL for 5 days to induce 3T3-L1 insulin resistance. We treated 3T3-L1 cells with TNFα and DHM (1 μM, Sigma Chemical Inc., Louis, MO, USA) for 5 days to demonstrate DHM ameliorated inflammation-induced insulin resistance. We treated 3T3-L1 cells with TNFα, DHM, and Compound C (the AMPK inhibitor, 5 μM, MedChemExpress, Monmouth Junction, USA) for 5 days to demonstrate DHM ameliorated inflammation-induced insulin resistance through AMPK. We treated 3T3-L1 cells with TNFα, DHM, and STO-609 (the CaMKK inhibitor, 10 ng/mL, MedChemExpress, Monmouth Junction, USA) for 5 days to demonstrate DHM ameliorated inflammation-induced insulin resistance through CaMKK. We treated 3T3-L1 cells with TNFα, DHM, and U73122 (1 μM, MedChemExpress, Monmouth Junction, USA) for 5 days to demonstrate DHM ameliorated inflammation-induced insulin resistance through the PLC-IP3 receptor pathway.