Differentiation of Motor Neuronal and Muscle Cell Models

NSC34 cells were used as motor neuronal cell model. Cells were routinely used in our lab^{11 (link),13 (link),30 ,31 (link)}. Briefly, cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) high glucose medium (Euroclone, Pero, MI, Italy) supplemented with 1 mM glutamine (Euroclone), penicillin (SERVA, Electrophoresis GmbH, Heidelberg, Germany) and streptomycin (SERVA) and 5% fetal bovine serum (Sigma-Aldrich). Cells were grown at 37 °C with 5% CO₂. To differentiate NSC34, retinoic acid 1 μM (Sigma-Aldrich) was added to full medium for 24 hrs before transfection and maintained for 72 hrs. C2C12 cells were used as muscle model. Cells were cultured as previously described^{32 (link)}, and maintained in DMEM high glucose medium completed with glutamine, penicillin, streptomycin and 10% of fetal bovine serum (GIBCO, Thermo Scientific Life Sciences Research, Waltham, MA, USA). Cells were grown at 37 °C with 5% CO₂. C2C12 were differentiated by supplementing medium with 2% Horse Serum (HS; Sigma-Aldrich), instead of 10% FBS, for 72 hrs.