Figure 4
From: Unscheduled expression of CDC25B in S-phase leads to replicative stress and DNA damage
Elevated level of CDC25B causes increased CDC45 recruitment on chromatin. (A) U2OS cells conditionally expressing CDC25B were induced (U2OS CDC25B) or not (U2OS) in the presence of 2.5 mM thymidine for 17 h then released from the cell cycle block by thymidine removal. After 3 h, the cells were harvested and processed for western blot analysis of two chromatin extracts, the detergent-soluble fraction (Sol. Chrm.) and the DNase1 soluble fraction (DNase). The CDC45 protein level was monitored using Orc2 as loading marker. A typical western blot of one of the four independent experiments used for quantification is shown. * indicates a p value < 0.01 (t-test where U2OS CDC25B is compared to U2OS). (B) U2OS conditionally expressing CDC25B were transfected with CDC45 or control siRNA (Scr) for 28 h or not transfected (NT) then treated with 2.5 mM thymidine for 17 h in the absence of tetracycline to induce CDC25B expression. Cells were released from thymidine block by thymidine removal from the media. After 3 h, total extracts were processed for western blot analysis using anti-Ha, anti-CDC45, anti-Îł-H2AX and anti-actin antibodies as loading controls. The CDC45 antibody was already tested for its specificity in [46]. Non-induced U2OS are shown as controls (U2OS).