Fig. 4. REF-52 cells remain arrested for at least 7 days following release from prolonged treatment with aphidicolin or hydroxyurea. (A) FACScan analysis of G0 synchronized REF-52 cells treated with aphidicolin (10 µM) for the indicated times and then released for 24 hours or for 7 days in nocodazole (0.25 µg/ml). Cells do not replicate and progress to mitosis during this period of time, as shown by the absence of 4N population. (B) Cell counts confirm the absence of cell proliferation after aphidicolin treatment for times equal to or greater than 45 hours. G0 synchronized REF-52 cells treated with aphidicolin for the indicated times were either harvested (time 0) and counted or were released into drug-free medium. Cell counts were then taken every 24 hours over 4 days in the released population. For counting, cells that had initially been plated at equal density were trypsinized, resuspended in 1 ml PBS and the number of cells present in 20 µl was determined using a Thoma cell counting chamber. Each value is an average of at least 8 counts, and error bars represent the corresponding standard deviations.

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