Fig. 7. DNA replication is suppressed in vivo following prolonged aphidicolin exposure, but extracted nuclei remain competent for replication. (A) In vivo analysis of DNA replication. Aphidicolin (10 µM)-treated REF-52 cells were released for 30 minutes in drug-free medium containing 10 µM BrdU and the percentages of replicating cells double-labeled for BrdU and for propidium iodide were determined by confocal immunofluorescence microscopy. An average of 1000 cells were counted in each of at least three samples at each time point. Values represent the percentages of BrdU-positive and -negative cells. Error bars represent the corresponding standard deviations. (B) Quantitative analysis of in vitro replicated nuclei derived from REF-52 cells and counts derived from microscopic imaging, as in part C. Following 15 hours of exposure to aphidicolin almost no nuclei (less than 1%) are positive. After 30 hours and 60 hours of exposure prior to harvest of nuclei, positive nuclei are 52% and 54% of the total, respectively. This experiment was replicated three times with similar results. An average of 450 nuclei were counted for each condition. (C) Visualization of in vitro DNA replication. REF-52 cells were released from G0 in the presence of aphidicolin (10 µM) and nuclei were harvested at the times indicated. Isolated nuclei were then subjected to in vitro DNA replication protocol as described in Materials and Methods. Fields of nuclei are shown following the replication assay, imaged for presence of biotin-16-dUTP and counterstained with propidium iodide. In the merged images green nuclei are biotin-16-dUTP positive and red nuclei are positive for only propidium iodide.

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