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. 2014 Feb 25;289(16):11374–11384. doi: 10.1074/jbc.M113.531707

FIGURE 6.

FIGURE 6.

Analysis of DNA replication elongation in the presence of CPT. A, schematic representation of the DNA fiber assay in the presence CPT. Cells were pulse-labeled with CldU (red) and successively pulse-labeled with IdU (green) in the presence of 100 nm CPT. To unify the encounter rate between the replication fork and the Top1 cleavage complex induced by CPT, pulse labeling time with CldU or IdU was doubled in TIPIN KO cells compared with wild-type cells. B, image of typical DNA fibers following CPT treatment. Cells were pulse-labeled first with CldU (red) and then IdU (green) in the presence of CPT. Unimpeded or impeded replication-reflected DNA fibers are shown. The CldU/IdU ratio indicates the value of the CldU-labeled fiber length divided by the IdU-labeled fiber length. C, histograms of the CldU/IdU ratio. The CldU/IdU ratio was calculated as the CldU-labeled fiber length divided by the IdU-labeled fiber length. p values were calculated by Student's t test. N, number of measured fibers. n.s., not significant.