Culture of L6-GLUT4myc Cells and Transfection

L6-GLUT4myc cells (25) were obtained from Kerafast and cultured according to the provider’s specifications. For experiments, L6-GLUT4myc myoblasts were plated at a concentration of 4 × 10⁴ cells/mL in 24-well plates. On the following day, the growth medium was replaced with antibiotic-free α-minimum essential medium Eagle (α-MEM) containing 20% FBS, and cells were transfected with a plasmid coding for hM3Dq (pcDNA3-HA-hM3Dq-p2A-mCitrine; University of North Carolina at Chapel Hill Vector Core) or pCMV-GFP (plasmid #11153; Addgene) using Lipofectamine 3000 (Thermo Fisher Scientific). About 24 h later, the medium was replaced with the original complete growth medium (α-MEM). Experiments were performed on the second day after transfection following serum starvation of cells for 3 h.