Figure 1

PAX3-FKHR regulates CCN3 gene expression in muscle cells. (a) Detection of secreted CCN3 protein in proliferating (GM) and differentiated (DM) C2 myoblast cells, aRMS (RH4, RH30) cells, and C2 cells stably expressing PAX3-FKHR (GM). For C2 cells, proliferating cell media (GM) were collected 24 h after plating, and differentiated cell media (DM) were collected 24 h after 2-days of differentiation. For aRMS cells, proliferating cell media were collected 72 h after plating without media change during this period. The results from C2 samples cannot be directly compared with the aRMS samples because of differing collection times. RIPA extract was prepared from cells for myosin heavy chain (MHC) and α-tubulin detection. (b) siRNA-knockdown of PAX3-FKHR reduced CCN3, but not CCN2 protein expression in RH4 cells. Transfected cells were supplemented with new media 24 h after transfection. Culture medium was collected 48 h later for CCN3 and CCN2 detection by western blot. RIPA extract was prepared from cells for PAX3-FKHR and α-tubulin detection. (a, b) Heparin-agarose samples were normalized as described in Materials and methods.