C2C12 myotube culture and Pi treatments

Mouse C2C12 myoblasts seeded into 6-well plates at a density of 2x10⁵ cells/well were first cultured for 3 days in DMEM/HG [Dulbecco's modified Eagle medium/high glucose (4500 mg/L)] supplemented with 10% fetal bovine serum (FBS, Gibco) at 37°C and 5% CO₂. They were then cultured for an additional 4 days in differentiation medium consisting of DMEM/HG supplemented with 2% adult horse serum (HS, Gibco). During this period the spindle-shaped C2C12 myoblast cells differentiated and fused, gradually forming multinucleate myotubes. The myotubes thus formed were then incubated for 24 h in sodium phosphate buffer (1M Na₂HPO₄/NaH₂PO₄, pH 7.4) titrated to achieve final Pi concentrations of 0, 0.5, 1, 2, 3, or 4 mM. Thereafter, the myotubes were harvested or lysed for downstream analyses. A minimum of three independent experiments were performed for each condition.