Figure 4.
MyoD protein levels are reduced and E-box–luciferase activation is blunted in GFP+mut 3′-UTR cells after 24 h in differentiation media. (A) The pattern of MyoD expression in control C2C12, GFP+wt 3′-UTR pool, and GFP+mut 3′-UTR pool cells cultured in differentiation media was analyzed by Western blots. A representative Western blot is shown, and the graph reports the average behavior of MyoD expression in each cell population, as determined by quantifying results from five independent experiments. MyoD levels are normalized to dynein levels, which do not change during differentiation, and are analyzed relative to the levels measured in proliferating cells (0 h in differentiation media). The difference in the MyoD expression pattern between GFP+mut 3′-UTR pool cells and both C2C12 and GFP+wt 3′-UTR pool cells is statistically significant (P < 0.05) only after 24 h (*). (B) A parallel analysis of the Myf5 expression pattern revealed no statistically significant differences at any time point between the GFP+mut 3′-UTR pool and the control C2C12 and GFP+wt 3′-UTR pool populations. (C) A luciferase (LUC) reporter construct containing three E-boxes was used to measure E-box–mediated gene expression in control C2C12, GFP+wt 3′-UTR pool, and GFP+mut 3′-UTR pool cells. The graph shows the average fold induction of E-box–luciferase expression relative to luciferase levels in proliferating cells (from three independent experiments). All error bars represent one standard deviation.