Figure 4.

PARPis induce varying degrees of replication fork speed on UWB-OlaJR. DNA fiber assays were performed to study replication fork dynamics. The schematics protocol and DNA fiber images of representative strands are shown. Representative DNA fibers were visualized by immunostaining of CldU (green) and IdU (red). The concentrations for each PARPi used are detailed in the Section 2. (A) For measuring replication fork speed, cells were cultured with CldU followed by IdU for 30 min each with or without PARPis. Replicating 1 µm roughly corresponds to 2.59 kb. (B) For measuring replication fork protection, cells were cultured with CldU and IdU for 20 min, followed by treatment with 2 mM HU with or without PARPis. The ratio of IdU-labeled tracts to CldU was calculated to determine the degradation of nascent DNA. All experiments were repeated in triplicate. Data are shown as mean ± SD. ****, p < 0.0001; ***, p < 0.001; ns, not significant.