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Figure 4


Fig. 4. Localization of EBNA1 dots in G2-phase-enriched cells. (A) Cells harboring the recombinant EBV were synchronized at the G1-S boundary by first thymidine and then aphidicolin block. Cells were then released by removing aphidicolin, and cell cycle progression was monitored by FACS analyses. (B) Immunofluorescence images (stained by anti-HA antibody) of two representative interphase cells that were harvested at 6 hours after release. Closely spaced double-dots of EBNA1 (red signals) are indicated by arrowheads. (C) Immunofluorescence images of two representative G2-PCCs (out of more than 100 acquired images of G2-PCCs). Calyculin A-treated cells were harvested at 6 hours after release. Note that there are many symmetrical double dots on sister chromatids (arrowheads). (D) Quantitative analysis to determine the frequency of symmetrical EBNA1 doublets in individual cells. The signals of EBNA1 (red) and CENP-C (green) are shown. Number of paired EBNA1 dots with paired CENP-C dots are assumed to be symmetrically localized EBNA1 dots (surrounded by a red rectangle). Number of symmetrically localized EBNA1 dots are indicated in each sub-panel. Additional EBNA1 double dots that do not fit the above criteria are also shown. Question marks indicate obscure CENP-C staining. Bars, 5 µm.





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