Figure 7.

p18^Hamlet downregulation prevents differentiation of C2C12 myoblasts, and its overexpression is sufficient to induce muscle-specific gene expression. (A) C2C12 myoblasts were transfected with control or two different p18^Hamlet siRNAs, and 48 h later were seeded at 70% of confluency and incubated for up to 3 days in DM. Expression levels of p18^Hamlet, myogenin and tubulin proteins were analysed by immunoblotting. (B) RNA was purified from C2C12 myoblasts that were transfected with control or p18^Hamlet-oligo 1 siRNAs and then maintained in DM for the indicated times or in GM. Myogenin (early gene), and MCK and MHC (late genes) mRNA levels were analysed by qRT–PCR. GAPDH mRNA levels were used for normalization. The histogram shows values of triplicate qRT–PCR reactions. Error bars represent s.d. Each experiment was performed at least twice with independent RNA samples and yielded similar results. (C) Downregulation of p18^Hamlet abrogates the formation of the characteristic multinucleated muscle fibres (upper panel). Bar is 250 μm. Formation of muscle fibres was confirmed by immunofluorescence with MHC antibodies. White arrowheads indicate the presence of multiple nuclei (stained with DAPI) in each fibre (lower panel). Bar is 10 μm. (D) C2C12 myoblasts were transfected with a p18^Hamlet expression vector and incubated with G418 to select clones (see Figure 4C). The mRNA levels of the indicated early and late muscle differentiation markers were analysed by qRT–PCR and normalized to GAPDH. Average values of triplicate qRT–PCR are shown. Error bars represent s.d.