Cells were pulse-labeled with 25 μM CldU (Sigma-Aldrich, C6891mg) and 250 μM IdU (Sigma-Aldrich, I7125-5g) at indicated times, with or without treatment, as reported in the experimental schemes. DNA fibers were prepared as previously reported (27 (link)). For the immunodetection of labeled tracks, the following primary antibodies were used: anti-CldU [rat monoclonal anti–5-bromo-2′-deoxyuridine (BrdU)/CldU; BU1/75 ICR1, Novus, 1:100] and anti-IdU (mouse monoclonal anti-BrdU/IdU; clone B44, Becton Dickinson, 1:50). The secondary antibodies were goat anti-mouse Alexa Fluor 594 (Abcam, ab150116, 1:200) or goat anti-rat Alexa Fluor 488 (Abcam, ab150157, 1:200). The incubation with antibodies was accomplished in a humidified chamber for 1 hour at room temperature. Images were acquired randomly from fields with untangled fibers using a Nikon Eclipse 80i fluorescence microscope, equipped with a video confocal system. The length of labeled tracks was measured using Fiji software, and values were converted into kilobases using the conversion factor of 1 μm = 2.59 kb. A minimum of 100 individual fibers was analyzed for each experiment, and the mean of at least three independent experiments is presented. Statistical analysis was performed using GraphPad Prism Software.