Mouse c2c12 myoblasts

For co-culture experiments, 25 × 10³ mouse C2C12 myoblasts (Sigma–Aldrich, St. Louis, MO, USA) and 25 × 10³ of rADSCs were seeded on sterile coverslips and cultured in high-glucose DMEM supplemented with 10% of FBS and 10 μg/mL gentamycin, in the presence or absence of IL-4 or SDF-1 for 7 days. Rat ADSCs used for the co-culture were first labelled with BacMam GFP Transduction Control (Invitrogen), according to the manufacturer’s instructions. Culture medium was replaced every day to keep required levels of cytokines. Cells were fixed with 3% paraformaldehyde (PFA) and subjected to immunolocalizatio analysis. BacMam GFP allowed for the identification of hybrid myotubes, i.e., ones generated by rADSCs fusing with C2C12 myoblasts. Nuclei were stained with Hoechst 33,342 (Sigma Aldrich) or DRAQ5 (Biostatus Limited), diluted 1:1000 in PBS. In selected experiments, cells were stained with either anti-myosin (skeletal) antibody (M7523, Sigma Aldrich) or anti-skeletal muscle marker (12/101-c, DSHB) to visualize the localization of myotubes in the culture. For each experimental group, the number of hybrid myotubes was counted from at least five fields of view of three independent experiments.

Mouse C2C12 myoblasts (Sigma-Aldrich) were maintained in Dulbecco’s modified Eagle medium (DMEM, Sigma-Aldrich) supplemented with 20% foetal bovine serum (FBS) and 1% penicillin/streptomycin (Sigma-Aldrich); referred to as growth medium or GM. To induce differentiation, the cells were seeded into µ-Plate 96 Well (Ibidi GmbH, Gräfelfing, Germany) at 2.8 × 10⁴ cells per well and incubated at 37 °C, 5% CO₂ overnight. The next day, the cells were washed three times with Hank’s Balanced Salt Solution (HBSS, Sigma-Aldrich), and DMEM supplemented with 2% horse serum and 1% penicillin/streptomycin (differentiation medium; DM) was added. Differentiation was allowed to proceed for 3–5 days, at which point cell fusion was assessed. All drugs were prepared in working stocks of DMSO, and the final concentration of DMSO in all treatments was 0.1%.