DNA Fiber Assay for Replication Dynamics

DNA fiber assay was performed as previously described [25 (link)] with the following modification: 1.5 × 10⁵ A2780 and A2780Cis cells were seeded into 6-well plates. The following day cells were synchronized at the G1/S boundary by 6 µM aphidicolin for 24 h [26 (link)]. Cells were washed 2 times with warm PBS, one time with warm RPMI media, and treated as indicated. During the last hour of drug treatment, cells were labeled with CldU 25 μM (Sigma-Aldrich, C6891) for 30 min and then with 250 μM IdU (Sigma-Aldrich, #I7125) for 30 min. Cells were harvested by trypsinization, resuspended in PBS, and samples were diluted to approximately 7 × 10⁵ cells/mL. Fiber spreading, denaturation, fixation, and staining were performed in ibiTreat µ-slide VI 0.4 (Ibidi, #80606). Fluorescence images were captured using a NikonTi2 fluorescence microscope with the 40× objective (with MilliQ water) with excitation wavelengths of 488 and 568 nm, and analyzed using the ImageJ software. At least 100 unidirectional forks labeled with both CldU and IdU were measured for every condition using Fiji [27 (link),28 (link)]. Antibodies used are listed in Table S1.