Fig. 6. Tyramide-coupled fluorescence in situ hybridization (T-FISH) of human papillomavirus (HPV) replicons. E2 is required for viral origin DNA association with spindles during metaphase. COS7 cells were transfected with the viral pMT2-H11URR-EEE1 (A) or pMTX-H11URR-EEE1-E2 (B). Twenty hours after electroporation, 100 m M 17b -estradiol was introduced for 4 h. Cells were fixed, and T-FISH was conducted with nick-translated pMT2-11URR-EEE1 as a probe (A Left and B Left). b -Tubulin was then probed with an antibody (Cy-3) (Center). A Right and B Right display merged images in which chromosomal DNA was stained with 4',6-diamidino-2-phenylindole (blue).