Figure 2

Shh increases C2 myoblast differentiation. C2 cells were exposed to ShhQT6 conditioned or control QT6 conditioned medium during both growth and differentiation. A. C2 myoblast lines C2/4 and C2C12 show enhanced differentiation in response to Shh. B. Quantitative comparison of enhanced differentiation of C2 myoblast lines in response to Shh calculated as the number of nuclei within MyHC-containing cytoplasm (i.e. number of differentiated myocytes). Note that the effect of Shh appears more dramatic if the proportion of nuclei in MyHC-containing cytoplasm is measured, because Shh-treated wells have the same or fewer total cells. For example, in one experiment C2/4 cells showed around 44% differentiation compared to ~6% in controls although across all experiments cell numbers were not significantly affected by Shh treatment. C. BrdU staining of C2/4 cells treated with ShhQT6 or control QT6 conditioned growth medium prior to a 2 hour BrdU pulse. D. In situ mRNA hybridisation on C2/4 myoblasts treated for one day with ShhQT6 conditioned growth medium revealed increased reactivity with a Gli1 antisense, but not sense probe, compared to myoblasts exposed to conditioned medium from control QT6 cells. E. Immunocytochemical analysis of C2/4 myoblasts similarly exposed to ShhQT6 conditioned or control QT6-conditioned growth medium using antibodies to MyoD, Myogenin and pan-MyHC.