shRNA knock-down and rescue experiment

Short hairpin RNA(shRNA) plasmid for the depletion of myogenin (Myog) were designed by the RNAi consortium in the pLOK.1 plasmid (Open Biosystems) as previously described [31]. At least three constructs targeting murine Myog and one scrambled control were linearized using the ScaI restriction enzyme (New England Biolabs), transfected into C2C12 cells or 10T1/2 cells, and the stable colonies are selected with puromycin (2 μg/ml). Individual clones were selected and propagated, and confirmed by mRNA and protein analysis.

For MyoD rescue in C2C12 cells or 10T1/2 stable cell line generation experiment, pEF6-MyoD plasmids transfected in the respective cell lines using Turbofect (Fisher Scientific) transfection reagent as previously described. The stable clones were selected with blasticidin (10μg/ml), clones were confirmed by mRNA and protein analysis.