Unknown

L8 myoblasts were grown in growth medium (15% bovine calf serum in Dulbecco’s modified Eagle’s medium). For induction of differentiation, the medium was changed to differentiation medium (no serum, 10 mkg/ml insulin, and transferrin in Dulbecco’s modified Eagle’s medium) when the culture became confluent. JX401, and its various derivatives were dissolved in dimethyl sulfoxide (DMSO) to a concentration of 10 mM. Aliquots from these stocks were added directly to differentiation medium to obtain a final concentration of 10 mkM. Control cells were incubated with the same volumes of DMSO without the inhibitors. The growth medium was replaced, and fresh inhibitors were added every 24 h. Preparation of lysates of L8 cells was performed as described previously. For monitoring myogenin induction, 40 mkg of lysates were separated by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose and probed with anti-myogenin monoclonal antibodies.