Versikine Fragment Induces Myoblast Differentiation

HEK293T cells were grown in Dulbecco’s Modified Eagle Medium (DMEM; 25 mM glucose) containing 10% fetal bovine serum (FBS) in atmospheric O₂ and 5% CO₂ at 37 °C. Cells were transfected using Lipofectamine 2000 (Thermo Fisher Scientific) with constructs encoding the V1 versican construct (kindly provided by Dieter Zimmermann), the bioactive G1-DPEAAE versikine fragment was produced by the insertion of a stop codon in the V1 versican construct after the Glu⁴⁴¹-Ala⁴⁴² peptide bond cleavage site [32 (link)], and empty vector control (pcDNA3.1MycHisA+ (Thermo Fisher Scientific)). Serum-free conditioned medium was collected for use in the myoblast differentiation experiments, as previously described [9 (link)], and underwent western blotting for confirmation of V1 versican and versikine protein expression. C2C12 myoblasts, a well characterized in vitro model of myoblast fusion and regenerative myogenesis [96 (link),97 (link)], were maintained in growth medium (25 mM glucose DMEM plus 10% FBS) in atmospheric O₂ and 5% CO₂ at 37 °C.