Cell culture and intervention protocols
C2C12 murine myoblasts (American Type Culture Collection, CRL-1772) were cultured in growth medium consisting of Dulbecco’s Modified Eagle’s Medium (DMEM; Thermo Fisher Scientific, USA) supplemented with 1% penicillin/streptomycin (100 units/mL penicillin and 100 µg/mL streptomycin; Thermo Fisher Scientific, USA) and 10% fetal bovine serum (FBS; Thermo Fisher Scientific, USA). Cells were cultured in a T-75 culture flask at a density of 5 x 103 viable cells/cm2 and were passaged at 70-80% confluence. Flasks were kept in a humidified incubator at 37°C with the supplementation of 5% CO2. To induce differentiation, confluent monolayers of C2C12 myoblasts were cultured in differentiation medium (DM) consist of DMEM supplemented with 1% penicillin/streptomycin and 2% horse serum (Thermo Fisher Scientific, USA) and the DM was changed daily. All experiments were performed on day 6 when most myoblasts have fused to form mature myotubes.
The gas phase of cigarette smoke, otherwise known as cigarette smoke extract (CSE), was prepared by bubbling 1 cigarette (Winfield Red, Phillip Morris International, Australia) through 25 mL of pre-warmed DM at a rate of 5 mL·sec-1 to produce 100% CSE stock solution. The stock solution was sterile filtered and serially diluted with pre-warmed DM to obtain concentrations required for experimentation. Hydrogen peroxide (H2O2; Chem-Supply, Australia) was prepared in sterile water resulting in a 3,000 µM stock solution. The stock solution was serially diluted with pre-warmed DM to obtain the required concentrations for experimentation. Apocynin (Sigma-Aldrich, USA) was dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich, USA) to give a stock solution of 500µM (stock solution). The stock solution was diluted with pre-warmed DM to give a final administering concentration of 500nM (68). To ensure bioavailability, apocynin was pre-incubated for 30 min prior to administration of the respective oxidative insults (H2O2 or CSE), recapitulating that in our animal model.