Cell culture
C2C12 mouse myoblast cell lines were obtained from ATCC, CRL-1772) and
were allowed to grow in 90% Dulbecco’s modified high glucose Eagle’s
medium (DMEM) containing 10% v/v fetal bovine serum (FBS) in 10cm cell
culture dish at 37°C in 5% CO2 incubator until 80%
confluent. The media was replaced to myogenic differentiation medium
containing 98% DMEM and 2% v/v horse serum and was changed every other
day for 7 days. The differentiated myotubes were then plated in 96, 6
and 12 wells plates for RT-PCR and immunoblot analysis.