Figure 1

Effects of low level inducible PAX3-FKHR construct on transformation and growth suppression in NIH3T3 cells. (a) Western blot analysis of PAX3-FKHR-ER protein expression in pBabe and pK1 retroviral constructs. Lanes (loaded with 40 μg cell extract) containing vector control (C) and PAX3-FKHR-ER (PF-ER) in pBabe (pB) and pK1 transduced cells are labeled on the top, and positions of PAX3-FKHR-ER and actin loading control are labeled at the right. (b) Comparison of transcriptional activity of PAX3-FKHR-ER-transduced into NIH3T3 cells using pBabe and pK1 inducible systems. Dual luciferase assay was performed using PRS9 luciferase reporter with indicated concentrations of 4-hydroxytamoxifen (TMF). The luciferase activity was normalized for transfection efficiency and expressed as the mean (±s.d.) of triplicate assays. (c) Cell growth of NIH3T3 cells expressing PAX3-FKHR-ER in pBabe. Triplicate cultures of PAX3-FKHR-ER-transduced cells were treated with the indicated concentration of TMF, and counted at the indicated time points. The cell numbers were normalized to the first day counts and expressed as the mean (±s.d.) of triplicate assays. (d) Soft agar colony assay of PAX3-FKHR-ER-transduced NIN3T3 cells. Cells were incubated with the indicated TMF concentration in soft agar and scored after three weeks. Three plates were counted for each TMF dose and results expressed as the mean (±s.d.).