Ryanodine

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.

Ca²⁺ was measured by a Ca²⁺ fluorescent probe fluo‐4‐AM kit following the manufacturer's instructions. C2C12 cells were incubated with ryanodine (100 nM, MedChemExpress) or U73122 (1 μM, MedChemExpress, Monmouth Junction, USA) for 1 h to block the endoplasmic reticulum Ca²⁺ channel. Then, the cells were washed 3 times with Hank's balanced salt solution and incubated with 10 μM fluo‐4‐AM at 37℃ for 1 h. After incubation, cells were then rewashed 3 times. Nikon Eclipse Ti‐s microscopy (Nikon) was used to observe fluorescence. Fluorescent data were acquired after excitation at 490 nm and intensity at 525 nm.

Ca²⁺ was measured by a Ca²⁺ fluorescent probe fluo-4-AM kit following the manufacturer's instructions. 3T3-L1 cells were incubated with ryanodine (100 nM, MedChemExpress, Monmouth Junction, USA) or U73122 (1 μM, MedChemExpress, Monmouth Junction, USA) for 1 h to block the endoplasmic reticulum Ca²⁺ channel. Then, the cells were washed 3 times with Hank's Balanced Salt Solution and incubated with 10 μM fluo-4-AM at 37°C for 1 h. After incubation, cells were then rewashed 3 times. Nikon Eclipse Ti-s microscopy (Nikon, Tokyo, Japan) was used to observe fluorescence. Fluorescent data were acquired after excitation at 490 nm and intensity at 525 nm.