Primary MEFs were isolated from embryos as described previously (Tommasi et al. 2005 (link); Yoon et al. 2015 (link), 2019b (link)). Cells were pulse-labeled with 25 µM IdU (Sigma) for 20 min and subsequently washed twice with PBS buffer, followed by treatment with cisplatin (60 µM) and 250 µM CldU (Sigma) for 30 min. DNA fibers were spread on glass slides, and slides were incubated in 2.5 M HCl for 90 min and then washed with PBS buffer. The slides were incubated in blocking buffer (5% BSA in 1× PBS) for 2 h. Primary antibodies—rat anti-BrdU antibody (Abcam) and mouse anti-BrdU antibody (BD Bioscience)—were diluted in blocking buffer and incubated for 1 h followed by extensive washing with PBS buffer. Secondary antibodies—goat anti-rat Alexa 594 (Thermo Scientific) and goat anti-mouse Alexa 488 (Thermo Scientific)—were applied for 30 min and slides were mounted with antifade gold mounting medium (Invitrogen). DNA fibers were analyzed using a Nikon eclipse fluorescence microscope and quantified using NLS-Elements AR software.