Figure 7.
APC/CCdh1 inhibits new origin firing in S-phase after prolonged HU treatment. (A) Non-target (NT) or Cdh1 siRNA were transfected to hTERT-RPE cells before synchronizing them in S-phase by single thymidine block. Cells were then treated with HU (or left untreated (Cs)) during the indicated time. Chromatin extracts were prepared and analysed by western blot with the indicated antibodies. Input: whole cell lysates. Lamin B was used as loading control. (B, C, D) Schematic of the labelling protocol (B, upper-right panel). hTERT-RPE cells were transfected with the indicated siRNA and synchronized in S-phase before CldU (250 μM) labelling. Whole cell lysates of S-phase synchronized untreated cells were harvested for western blot analysis with the indicated antibodies (B, upper-left panel). Cdk4 was used as loading control. CldU track length of 300 fibres from three independent experiments was measured. CldU track length distribution and statistical analysis are shown (B, bottom panel). Box and whiskers show Min, Max, Median and first quartiles. Values marked with asterisks are significantly different (unpaired t-test, n.s.: non-statistically significant, ****P < 0.001). The percentage of replication fork restart, stalled replication forks and new origin firing relative to total CldU labelled fibres is shown in the graphs (C). More than 1000 fibres from three independent experiments were counted in each condition. Means and standard deviation (bars) are shown. Values marked with asterisks are significantly different (paired t-test, n.s.: non-statistically significant, *P < 0.05, **P < 0.01). Representative images of each condition are shown (D).