Downregulation of PAX3-FKHR decreases anchorage-independent growth of Rh30 cells. (A) shRNA against PAX3-FKHR downregulates the level of PAX3-FKHR. Rh30 cells stably transfected with either shRNA against PAX3-FKHR (KD1 and KD2, two different clones generated from the same shRNA) or a control non-targeting shRNA (CON) were used to analyze PAX3-FKHR mRNA or protein levels by using real-time RT-PCR (left panel) or Western blotting (right panel), respectively. GAPDH was used as an internal control for RT-PCR experiments, and PAX3-FKHR mRNA levels in KD1 and KD2 were normalized to CON (PF mRNA fold-change). Actin was the equal loading control for Western blot experiments. M, molecular weight marker; PF, PAX3-FKHR (~99kD); FKHR (~70kD) was also indicated. (B) Downregulation of PAX3-FKHR decreases anchorage-independent growth of Rh30. Raw fluorescent units (RFUs) were used to represent colony numbers of Rh30 control clone (CON) and KD1; and the average of triplicate measurements is shown.