Figure 3

Effects of low expression of PAX3-FKHR paired box and homeodomain mutations on transformation in NIH3T3 cells. (a) Western blot analysis of mutant PAX3-FKHR-ER constructs in pBabe. Lanes (loaded with 40 μg cell extract) containing vector control (C), wild-type (W), G48S (G) and N269A (N) mutated PAX3-FKHR-ER-transduced cells are labeled on the top, and positions of PAX3-FKHR-ER (PF-ER) and actin loading control are labeled at the right. (b) Transcriptional activity of WT and mutated PAX3-FKHR-ER constructs in pBabe. The dual luciferase assay was performed using a luciferase reporter containing a CD19 (paired box-specific) or a P3 (homeodomain-specific) binding site. This reporter was transfected in NIH3T3 cells transduced with vector control (C) or wild-type (WT), G48S (G) and N269A (N) PAX3-FKHR-ER constructs in pBabe, and treated with the indicated TMF concentrations. The luciferase activity was normalized for transfection efficiency and expressed as the mean (±s.d.) of triplicate assays. (c) Soft agar colony assay of WT and mutated PAX3-FKHR-ER constructs in pBabe. NIH3T3 cells transduced with the corresponding constructs 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.). (d) Focus formation assay of WT and mutated PAX3-FKHR-ER constructs in pBabe. NIH3T3 cells transduced with the corresponding constructs were seeded with excess untransduced NIH3T3 cells in 100 mm dishes, and treated with the indicated concentrations of TMF. After 2 weeks, three plates were stained and counted for each TMF dose and results expressed as the mean (±s.d.).