FIG. 2.

Heterologous expression of d-Asb11 or h-Asb9 prevents terminal differentiation in the C2C12 model system of skeletal muscle differentiation. (A) Schematic illustration of the various stages of skeletal muscle differentiation as observed in the C2C12 model system and the associated expression of markers of muscle differentiation. (B) Following 96 h of exposure to differentiation medium, terminal differentiation of skeletal muscle, as assayed by luciferase activity driven by the MCK promoter, was inhibited by heterologous expression of either asb11 or h-Asb9. Transfection of a vector expressing only myc-tag (MT) or a HA-tag suppressor of cytokine signaling (SOCS) box deficient d-Asb11 (HA-d-Asb11ΔSOCS) showed no inhibitory effects. As a control for transfection efficiency, cytomegalovirus (CMV) promoter activity was used. Error bars show standard error of the mean (SEM) of 3 experiments. Statistical testing was done by comparing experimental groups to MT-only-transfected cells using a heteroscedastic paired Student's t-test. (C) C2C12 cells were transfected with a vector containing only the MT, MT-asb11, HA-asb11ΔSOCS, or MT-hAsb9 and allowed to differentiate for 72 h in the appropriate medium. Positive expression of the constructs was assessed and is shown on the left side of the panel. Effects of these transgenes on the expression of myogenin (MyoG; a myoblast marker) and myosin heavy chain (MHC; a marker of terminal muscle cell differentiation) were tested either in undifferentiated cells (left) or after 72 h under differentiation medium treatment (right). A representative image from 3 independent experiments is shown. **P<0.01.