HU activates dormant origins within active replication factories. U2OS cells were synchronized in early S phase as described in Fig. 1. (a–c) Cells were transfected with GFP-PCNA 24 h before synchronization in early S phase. After HU treatment, cells were transfected with Cy3-dUTP and fixed for 30, 60, 90, or 120 min to analyze the percentage of colocalization between Cy3-dUTP (red) and GFP-PCNA foci (green). (a) Labeling scheme and representative images are shown. The percentage of colocalization within individual cells was calculated by dividing the colocalized volume of Cy3 and GFP-PCNA foci by the total volume of GFP-PCNA foci. Analysis of >40 cells gave rise to the mean percentage of colocalization at each time point and SEM. (b) Lines were fitted to the data points to calculate the gradient, which indicates the rate of replication within the factories. (c) Composite data from three independent experiments were combined to give a mean gradient and SEM between the three experiments. (d and e) U2OS cells were transfected with MCM5 siRNA 72 h before synchronization in early S phase. (d) Chromatin-bound MCM2 and MCM5 levels were determined by immunoblotting after MCM5 siRNA. After 200 µM HU treatment, cells were transfected with Cy3-dUTP and fixed for 30 or 120 min afterward. (e) The percentage of colocalization between Cy3-dUTP and PCNA foci (as revealed by anti-PCNA immunofluorescence) is shown.