Satellite Cell Culture and HDAC4 Deletion

C2C12 cells were grown in Dulbecco’s modified Eagle medium supplemented with 20% fetal bovine serum (Sigma-Aldrich), 2 mM glutamine (Sigma-Aldrich), 50 μg/ml gentamicin (Sigma-Aldrich) (GM). After one day, the medium was replaced with differentiation medium (DM) made of Dulbecco’s modified Eagle medium supplemented with 2% horse serum (Sigma-Aldrich), 2 mM glutamine (Sigma-Aldrich), 50 μg/ml gentamicin (Sigma-Aldrich).
Satellite cells were plated on 0,01% collagen (Sigma-Aldrich)-coated dishes with Dulbecco’s modified Eagle medium supplemented with 20% horse serum (Sigma-Aldrich),100 U/ml penicillin (Sigma-Aldrich), 100 μg/ml streptomycin (Sigma- Aldrich), 50 μg/ml gentamicin (Sigma-Aldrich), 3% of chicken embryo extract as growing medium (GM). After 3 days, the medium was replaced with differentiation medium (DM) (GM diluted 1:10).
To obtain HDAC4 deletion in vitro, satellite cells derived from HDAC4^fl/fl Pax7^CE/+Cre+ mice were treated with 0.4 µM 4 OH-TMX (Calbiochem) or vehicle (methanol) as controls, for 72 hours, as described in^{35 (link)}.
To inhibit P21 expression, satellite cells were treated with 10 µM UC2288 (trans-1-(4-chloro-3-trifluoromethyl-phenyl)-3-(4-hydroxy-cyclohexyl)-urea; Calbiochem) or vehicle (DMSO, Sigma-Aldrich), for 24 h hours, as described in^{51 (link)}.