C2C12 culture and differentiation

C2C12 murine myoblasts were provided by Dr. P. Porporato, University of Turin, Italy. Unless differently specified, all reagents were obtained from Sigma-Aldrich, Inc.; SDS-PAGE materials were from Bio-Rad Laboratories; anti-Myosin Heavy Chain (MHC) and anti-actin primary antibodies were from Santa Cruz; Alexa 488 fluorescent secondary antibodies were from Pierce. ECL detection reagents were from Bio-Rad Laboratories.

Murine C2C12 myoblasts were cultured in growing medium composed of Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum in 5% CO₂ humidified atmosphere. For differentiation, sub-confluent C2C12 were shifted from growing to differentiating medium composed of DMEM containing 2% Horse Serum (HOS). Confocal analysis. C2C12 myoblasts were grown until sub-confluence on glass coverslips (used as control) or LCN and then differentiated for four days. Myotubes were washed with PBS and fixed in 3% paraformaldehyde for 20 min at 4°C. Fixed cells were permeabilized with three washes with TBST (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 0.1% Triton X-100), and then blocked with 5.5% HOS in TBST for 1 h at room temperature. Cells were then incubated with specific primary antibodies, diluted 1:100 in TBS (50 mM Tris–HCl, pH 7.4, 150 mM NaCl), overnight at 4°C. Cells were then washed once with TBST and once with TBST with 0.1% BSA and then incubated with Alexa 488 secondary antibodies (diluted 1:100) for 1 h at room temperature in TBST with 3% BSA. For the staining of nuclei 4′,6-diamidino-2-phenylindole (DAPI) 10 μM final, was added to TBST and the cells were treated for five minutes at room temperature. After extensive washes in TBST, cells were mounted with glycerol plastine. In all experiments emitted fluorescence was analyzed using a confocal fluorescence microscope (Leica TCS SP8).

Myoblasts were plated at the same density on each substrate and the day after, the differentiating medium was added. After four days of differentiation the nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI), 10 μM final, and visualized under a confocal fluorescence microscope (Leica TCS SP8).

The differentiation index has been calculated as the percentage of Myosin Heavy Chain positive cells above the total nuclei. The fusion index is the average number of nuclei in Myosin Heavy Chain positive cells (containing at least three nuclei) above the total nuclei. Myotube width was measured by ImageJ. Ten randomly chosen fields for each sample were analyzed and the average value was reported in the bar graph.

Cells were lysed for 20 min on ice in 500 μl of complete radio-immunoprecipitation assay (RIPA) buffer. Lysates were clarified by centrifugation, and total protein contents were obtained using Bradford assay (Bio-Rad Laboratories). 20 μg of total proteins for each sample were separated by SDS-PAGE and transferred onto PVDF membranes. PVDF membranes were incubated in 2% milk, probed with primary antibodies, and incubated with secondary antibodies conjugated with horseradish peroxidase. Quantification of bands was achieved using ImageJ software.