Matrine

Three C2C12 myotube atrophy models were established, according to previous studies (31 (link)–33 (link)). Briefly, C2C12 myoblasts were differentiated to myotubes by culturing in 2% horse serum at 37°C. Dex (National Institute for the Control of Pharmaceutical and Biological Products, Beijing, China) or TNFα (Novus Biologicals, LLC, Littleton, CO, USA) were then added to the media for 48 h at 100 µM and 50 ng/ml, respectively. For the third model, the myotubes were incubated for 48 h in CM consisting of 33% ‘cachexia liquid’ and 66% fresh DMEM with 2% horse serum; this CM was replaced every 24 h. The ‘cachexia liquid’ was acquired as follows; when CT26 cells reached 90% confluence, their culture medium was replaced with 2% horse serum (differentiation medium) and the supernatant was collected as ‘cachexia liquid’ after 48 h. For the investigation of myoblast differentiation and myotube atrophy, matrine (Shanghai EFE Biological Technology Co., Ltd., Shanghai, China) was added to the culture medium at 0.1 and 0.2 mM for 48 h at 37°C. For the signalling pathway investigation, 0.1 mM matrine was added for 48 h at 37°C and 10 nM wortmannin (MedChemExpress, Monmouth Junction, NJ, USA) was added to culture medium for 48 h at 37°C.