Figure 5. Checkpoint kinases inhibit new replication factory activation. Cells were transfected with GFP-PCNA 24 h before synchronization into early S phase as described for Fig. 1. After release from thymidine for 1 h, cells were pulse labeled with Cy3-dUTP followed by treatment with 200 µM HU for 2 h in the presence or absence of caffeine. (a) Representative images of nuclei fixed at 30 (i) or 120 min (ii) after Cy3-dUTP pulse. This shows that cells were in the same early timing stage throughout the experiment. (b) The number of Cy3 foci colocalized with GFP-PCNA foci (>10% volume overlap), Cy3 foci not colocalized with GFP-PCNA foci (<10% volume overlap; Cy3-only foci), and the GFP-PCNA foci not colocalized with cy3 foci (<10% volume overlap; PCNA-only foci) were quantified, and their mean value per cell was represented as yellow, red, and green bars, respectively. Analysis of >50 cells gave rise to mean and SEM (error bars).
Figure 5.

Checkpoint kinases inhibit new replication factory activation. Cells were transfected with GFP-PCNA 24 h before synchronization into early S phase as described for Fig. 1. After release from thymidine for 1 h, cells were pulse labeled with Cy3-dUTP followed by treatment with 200 µM HU for 2 h in the presence or absence of caffeine. (a) Representative images of nuclei fixed at 30 (i) or 120 min (ii) after Cy3-dUTP pulse. This shows that cells were in the same early timing stage throughout the experiment. (b) The number of Cy3 foci colocalized with GFP-PCNA foci (>10% volume overlap), Cy3 foci not colocalized with GFP-PCNA foci (<10% volume overlap; Cy3-only foci), and the GFP-PCNA foci not colocalized with cy3 foci (<10% volume overlap; PCNA-only foci) were quantified, and their mean value per cell was represented as yellow, red, and green bars, respectively. Analysis of >50 cells gave rise to mean and SEM (error bars).

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal