Fig. 3. (A) Cell cycle dependent expression in cultured C2C12 myoblasts of genes involved in muscle regeneration. (A) RNA analysis of adherent myoblasts (Mb), suspension cultures at 12, 24 or 48 hours (S12, S24, S48), replated cultures at 2-30 hours (R2-R30) and myotubes (Mt). c-met transcripts are expressed throughout the cell cycle but are downregulated during differentiation. HGF transcripts are undetectable in asynchronous or differentiated cultures but expressed in synchronized cells during arrest and early activation. M-cad and PEA-3 mRNAs show classic cell cycle dependence: suppression in G0 and activation in G1 (PEA-3) and G1-S (M-Cad). Ethidium bromide staining of rRNA [28S] indicates equal loading. Data are representative of three independent experiments. (B) Semi-quantitative RT-PCR analysis of CD34 expression. Compared with asynchronous myoblasts (Mb), relative levels of CD34 mRNA rise in suspension-arrested cells (S12, S60) and decline during cell cycle activation upon replating (R1, R12, R30). Primers used detect a region common to both splice variants of CD34 mRNA. Values (upper panel) represent the means±s.e.m. of duplicate assays for CD34 RNA normalized with respect to L7 control RNA for each sample, shown in the Southern blot (lower panel). Similar results were obtained with two independent time course experiments.