The effect of checkpoint inhibition on replication factories. Cells were transfected with GFP-PCNA 24 h before synchronization in early S phase, and the DNA damage checkpoint in these cells was inhibited by either CHK1 siRNA (transfected simultaneously with GFP-PCNA) or 5 mM caffeine. (a) Total cell lysate was immunoblotted for phospho-Chk1, Chk1, and H3. (b) U2OS cells were synchronized in early S phase as described for Fig. 1. After release from thymidine for 1 h, cells were treated with 200 µM HU for 2 h in the presence or absence of caffeine. The number of GFP-PCNA foci in each cell (>50 cells analyzed for each condition) was determined, with error bars representing SEM. (c and d) The rate of DNA replication within factories was measured by the protocol described in Fig. 3, but this time, comparing cells where the checkpoint was inhibited by Chk1 siRNA or caffeine. (c) Data from one set of experiments are shown. Analysis of >50 cells gave rise to the mean percentage of colocalization at each time point and SEM. Lines were fitted to the data points to calculate the gradient, which indicates the rate of replication within the factories. (d) Two independent experiments (n = 2) were performed to give rise to the mean gradient under each condition, and statistical significance is shown as SEM between the two gradients.