Fig. 1.

Over-expression of ZBP-89 enhances the myogenic program in C2C12 skeletal muscle cells.(A) C2C12 myoblast cells (MB) were plated at 1×10⁴/well, incubated in GM (DMEM plus 10% FBS) or differentiated to myotubes (MT) in DM (DMEM plus 1% FBS) plus either Ad-β-gal, Ad-empty vector or Ad-ZBP-89 for 24 and 48 h, and visualized using a light microscope at 20X magnification. Representative fields from 3 independent experiments are shown. (B) Western blot analyses of WCEs (50 μg) isolated from C2C12 cells as grown in Fig. 1A for 0, 12, 24, 48 and 96 h as described in Materials and methods. Antibodies used for immunoblotting (IB) are indicated at dilutions of 1:1000 and β-tubulin is included as a loading control. (C) qPCR analysis of endogenous ZBP-89 mRNA levels in MBs plated for 24 h and harvested or MTs switched to DM and harvested at the indicated times. Details of qPCR conditions are discussed in Materials and methods. The y-axis represents the relative ZBP-89 mRNA levels normalized to U6 expression. Results are graphed with MB set as 1x and are the average of three separate experiments performed in triplicate with bars representing the standard error of the means (S.E.). (D) Graphical analysis of cells infected with Ad-β-gal, Ad-empty or Ad-ZBP-89 and stained with Hoechst to determine the number of nuclei per muscle fiber. Results are from three separate, independent experiments. (E.) MBs, MTs and Ad-infected cells were co-stained with Hoechst and MHC and then visualized via fluorescence microscopy. Results are representative of three independent experiments. (F.) Quantitation of microscopy results from part E. Results are graphed as the percentage of Hoechst stained cells that also express MHC.