Overall origin initiation and active replication factories is reduced by HU or aphidicolin. (a–d) U2OS cells were treated with 0–500 µM HU or 0–0.1 µg/ml aphidicolin for 4 h before pulsing with 10 µM EdU for 30 min. (a and c, closed squares) Cellular EdU incorporation was detected by flow cytometry with mean and SEM calculated from three independent experiments. (a and c, open squares) DNA fiber analysis was performed on parallel samples to determine mean fork speed. The ratio of cellular EdU incorporation to fork speed (b and d) indicates the relative number of forks per cell. (e and f) U2OS cells were synchronized in early S phase by nocodazole shake off followed by incubation with thymidine for 12 h. Cells were released from thymidine for 1 h and treated with HU for 2 h. (e) The number of active replication factories was determined by either transfecting cells with GFP-PCNA 24 h before synchronization or pulsing cells with Cy3-dUTP and fixing after 30 min. Mean cellular foci number and SEM were derived from >50 cells. (f) Representative images of factories labeled with GFP-PCNA.