Figure 2.

Notch3 downregulation promotes RMS cell differentiation. (a) RD and RH30 cells cultured in complete medium (i.e., supplemented with 10% of fetal calf serum) were analyzed after 6 days of Notch3 or control (CTR) siRNA treatment. Representative immunofluorescence showing de novo expression of endogenous myosin heavy chain (MHC, green) in multinucleated fibers of Notch3 siRNA-transfected RD and RH30 cells (white arrows). Representative of three assays. (b) Western blotting showing de novo expression of Troponin I and β-actin (loading control) in Notch3 siRNA RD and RH30 cells treated as in (a). (c) Representative light microscopy pictures of RD and RH30 cells showing elongated multinucleated structures in Notch3 siRNA-treated cells treated as in (a). (d) Western blotting showing levels of Notch3^IC and HES1 (left) and Myogenin along with the phosphorylation of p38, Akt and mTOR (right) in RD and RH30 cells 24 and 48 h after CTR or Notch3 siRNA transfection. β-actin was the loading control. Representative of three independent experiments. (e) Western blotting showing levels of Notch3^IC and HES1 in RD and RH30 cells 24 and 48 h after CTR or Jagged1 siRNA transfection. β-actin was the loading control. Representative of three independent experiments