Figure 2

Temporal activation of CCN3 protein (a) and mRNA (b) expression following PAX3-FKHR induction in C2 myoblast cells. Total RNA was collected from DOX-induced C2 cells expressing vector or vector containing PAX3 or PAX3-FKHR cDNA. Levels of CCN3 transcript were determined by quantitative real time RT−PCR. GAPDH was used for normalization of the RT samples. Fold change ratio compared GAPDH-normalized CCN3 mRNA level from PAX3 or PAX3-FKHR-expressing cells to that from vector expressing cells, and was calculated using the equation 2 to the power -delta delta Ct. (c) CCN3 induction by PAX3-FKHR was specific to myogenic cells. C2, NIH3T3 and RD eRMS cells were transiently transfected with vector or vector expressing PAX3-FKHR. RIPA extract and culture media were harvested 48-h post-transfection for detection of PAX3-FKHR and CCN3 proteins, respectively. PAX3 expression vector was tested in parallel in C2 and NIH3T3 cells under the same conditions. Heparin-agarose samples were normalized as described in Materials and methods.