Fig. 6. Interspecies heterokaryon assay using the human osteosarcoma cell line F6, stably transfected with SHOX and the mouse fibroblast cell line NIH-3T3. After induction of SHOX expression, F6 cells were fused to NIH cells to make heterokaryons that were immunostained with anti-SHOX antibodies. (A) Phase contrast image of a representative heterokaryon with two larger F6 nuclei and one smaller NIH-3T3 nucleus (arrows). (B) Hoechst staining of the F6 and NIH-3T3 nuclei. Arrows indicate different nuclear staining for the F6 and NIH-3T3 nuclei that are shown in A. (C) Immunostaining using rabbit anti-SHOX antibodies (green). In more than 100 heterokaryons analyzed, not a single mouse nucleus showed positive staining for SHOX, indicating that once inside the nucleus, SHOX shows no shuttling between the nucleus and the cytoplasm. Arrows indicate positive SHOX staining only in F6, but not in NIH-3T3 nuclei.