FIG 7.

The replication defects associated with Pol κ loss can be restored by Chk1 ectopic expression. (A) Cells were transfected with control siRNA (Luc), siRNA targeting the 3′ UTR of Chk1 (Chk1-3′), or Pol κ (Polκ3′). At 48 h after transfection, asynchronous cells were pulse-labeled with EdU (10 μM) for 30 min. Nuclear EdU intensities were quantified in S-phase nuclei. Medians with 25% and 75% interquartile ranges are represented. ****, P < 0.0001; Mann-Whitney test. Number of values (nb values) and median values are indicated. (B) EdU intensity relative to control conditions was determined in 3 independent experiments, and the mean (±SEM) of medians relative to siLuc condition is presented. *, P < 0.05; **, P < 0.01; t test. (C) Schematic representation of experimental DNA fiber labeling. RKO cells were cotransfected with the indicated siRNAs and a vector expressing either GFP-empty, GFP-Polκ, or myc-Chk1. At 48 h after transfection, ongoing DNA replication forks were labeled with IdU (green tracks) for 20 min, treated with HU 1 mM for 1 h, and then labeled with CldU (red tracks) for 30 min. The lengths of CldU tracks were measured and the values of two independent experiments were pooled and plotted. The number of CldU tracks (nb values) measured and the medians of their lengths are indicated. Medians with 25% and 75% interquartile ranges are represented. ***, P = 0.001; ***, P < 0.001; Mann-Whitney test).