Figure 2

MLP silencing reduces muscle cell differentiation. (a and b) C2C12 cells were transfected with MLP-specific shRNA and non-targeting scramble plasmid (ctrl) and then differentiated for 72 h. MLP expression was measured in myoblasts and myotubes as described under Materials and Methods. α-Tubulin was used as loading control. Densitometric analysis was performed with ImageJ and average values from three separate experiments are reported below the bands. *Significantly different from ctrl myoblasts or myotubes. Significance was set at P<0.05. (c) Downregulation of MLP mRNA in MLP-silenced myoblasts and myotubes measured by real-time PCR. MLP mRNA from ctrl cells was set at 1. (d) MyoG1 and MyoD1 mRNA expression in myotubes from ctrl and shMLP cells measured by real-time PCR. MLP mRNA from ctrl cells was set at 1. (e) Images from ctrl and MLP-silenced cells differentiated as described under Materials and Methods. Formation of elongated myotubes is observed in ctrl cells but is missing in MLP-silenced cells in which many rounded cells are present. (f) Optical microscopy images of fixed and epon-embedded myotubes prior to electron microscopy analysis. During differentiation (72h), ctrl cells form multinucleated myotubes while MLP silencing cells have defective myotube formation with fiber-like disorganized structure. *Statistically significant from ctrl cells. Significance was set at P<0.05.