Figure 5
From: Unscheduled expression of CDC25B in S-phase leads to replicative stress and DNA damage
Elevated level of CDC25B causes perturbation of S-phase progression. (A) U2OS were induced to express Ha-CDC25B (U2OS CDC25B) or not (U2OS) then incubated in the presence of 2.5 mM thymidine for 17 h and released. Two hours after release, replication sites were pulse labeled for 30 min with CldU then for 30 min with IdU. After immunostaining with antibodies specific for CldU and IdU, the overlapping foci areas were quantified in each isolated cell and the results were expressed as the colocalization ratio (about 50 cells were analyzed). The insert shown in the central panel of this figure displays representative labeling with CldU and IdU and the typical merge figure that supports colocalization quantification using Image J software. (B) HCT116 cells expressing (CDC25B+) or not (parental) elevated levels of CDC25B (see figure 2) were processed after thymidine treatment as in (A). A total of 100 to 150 cells were analyzed. (C) HCT116 expressing elevated levels of CDC25B (HCT CDC25B+) were transfected with a CDC25B siRNA (siCDC25B) or a control siRNA (siScr) for 24 h then treated as in A and B. As controls, untransfected HCT116 CDC25B+ (NT) and parental cells (ctr). *** indicates a p value < 0.0001. A total of 130 to 170 cells were analyzed.