Focus-forming activity of TEL-FGFR3 and subcellular localization of TEL-FGFR3 chimeric protein in NIH/3T3 cells. (A) Representative results of the focus-forming assay in NIH/3T3 cells. NIH/3T3 cells were transfected with 30 μg of the indicated constructs and cultured for 2 weeks. Giemsa staining was performed for visualization of foci. (B) Comparison of the number of foci per dish. NIH/3T3 cells were transfected with 20 μg or 30 μg of the indicated DNA constructs. Colonies larger than 1 mm in diameter were counted. The results are expressed as the means plus SD of triplicate plates. Similar results were obtained in 2 independent experiments. (C) Subcellular localization of TEL-FGFR3 protein in NIH/3T3 cells by immunofluorescence staining. NIH/3T3 cells were transfected with (i) TF-V5, (ii) ΔHLH-TF-V5, or (iii) empty vector (mock), and (iv) nontransfected NIH/3T3 cells. Cells were cultured on microscope slides for 48 hours, fixed, and then subjected to permeabilization and indirect staining, as described in “Materials and methods.” DAPI was used as a nuclear counterstain. Expression of V5-associated protein derived from the EF-1 α promoter of TF-V5, ΔHLH-TF-V5, or mock and GFP-zeocin fusion protein derived from the independent cytomegalovirus (CMV) promoter in the same vectors was analyzed with fluorescence microscopy using the red (rhodamine), green (GFP), and blue (DAPI) channels. Images were captured with a CCD camera attached to a Nikon Eclipse E800 microscope with a Nikon Plan Apo 100×/1.40 oil objective lens (Nikon), and cropped and merged in Leica QFISH software (original magnification × 1000).