L6 rat skeletal muscle myoblasts

L6 rat skeletal muscle myoblasts were purchased from American Type Culture Collection. Cells were cultured in 10‐cm plates with growth medium (GM: AMEM supplemented with 10% FBS and 1% antibiotic‐antimycotic preparations) as described before (Moghei et al., 2016). Cells were seeded (2 × 10⁵ cells/well) in six‐well plates for western blot experiments or (10⁵ cells/well) in 12‐well plates for glucose transport experiments. They were allowed to proliferate for 48 hr or until they became 90–100% confluent. They were then shifted into differentiation medium (DM: AMEM, 2% HS, 1% antibiotic–antimycotic preparations) and replenished with fresh DM every 48 hr. Myotubes were used on d 5 or d 6 of differentiation.

L6 rat skeletal muscle myoblasts were obtained from the American Type Culture Collection. Cells were cultured in 6‐well plates (150–250 × 10³ cells/well) or in 10‐cm plates (600 × 10³ cells/plate). They were propagated at 37°C and 5% CO₂ in humidified atmosphere in proliferation medium composed of AMEM supplemented with 10% FBS and 1% Ab‐Am until they reached at least ~90% confluency. Cells were then either harvested (day 0) or shifted into differentiation medium (DM) (AMEM supplemented with 2% HS and 1% Ab‐Am). They were harvested day 1–5 during differentiation. Differentiation medium was replaced with fresh medium every other day. To ascertain that our observations were not limited to L6 cells, some of the experiments were also carried out in C2C12. In such experiments, C2C12 were cultured as above but in DMEM growth medium (DMEM, 10% FBS, 1% Ab‐Am). For differentiation, cells were cultured in DMEM‐based differentiation medium (DMEM, 2% HS and 1% Ab‐Am).