*3.9. Myogenic Di*ff*erentiation*

C2C12 myoblasts were seeded onto six-well plates (4 × 10<sup>4</sup> cells/well) in triplicate and cultured for 2 days in DMEM. Myogenesis was induced by culturing cells in differentiation medium (DMEM, 2% Horse serum, 1% P/S) containing PN at 0.5, 1 μg/mL for 5 days [35]. Differentiated muscle cells were immunostained for fast myosin heavy chain (f-MyHC, a marker of terminal myogenic differentiation), fixed with 4% paraformaldehyde, blocked with 5% goa<sup>t</sup> serum, and incubated at room temperature with myosin 4 monoclonal antibody (MF20)-Alexa Fluor 488 (Thermo Scientific) overnight at 4 ◦C. The nuclei were visualized by staining with DAPI (Sigma) for 10 min. Total cell nuclei and nuclei within f-MyHC-positive myofibers in at least 15 fields of three replicate platings were counted by using ImageJ. Myotube fusion indices were calculated by expressing the number of cells expressing f-MyHC (green) as a percentage of total nuclei (DAPI, blue) [35,36].
