Figure 4.

Fluorescence microscopy analysis of cell fusions. (A) Centrosome analysis in cell fusions. Wild-type U2OS cells were stained with CellTracker Blue and fused with GFP-NEDD1–expressing U2OS cells. Fused cells were fixed in methanol and stained for γ-tubulin (red). (i) Heterotypic fused cells could be identified by blue and green cytoplasmic fluorescence, with homotypic fusions showing (ii) only blue or (iii) only green fluorescence. The CellTracker Blue dye mixed in (the cytoplasm) fused cells but was not transferable between adjacent unfused cells. Staining with γ-tubulin antibodies revealed that GFP-NEDD1 incorporates into the centrosomes of the wild-type (nonexpressing) fusion partner. (B) Limitation of DNA damage signal to irradiated fusion partners. Cells were untreated or irradiated with 5 Gy and then fused with the indicated partners. Fixation and staining were performed as described in A, with antibodies to γ-H2AX (red) in place of those for γ-tubulin. (C) Quantitation of γ-H2AX signal in nuclei of untreated and 5-Gy-treated cells fused to irradiated partners. Representative example of experiment in which at least 100 cells were scored.