DNA fiber assay

The progression of replication forks after cisplatin and/or VE-821 (ATR inhibitor) treatment was evaluated by DNA fiber assays; for this, MCF-7 cells were exposed to a 20-min pulse of 20 µM CldU, followed by co-incubation (60 min) with 200 µM IdU and cisplatin (150 and 300 μM) and/or VE-821 (1 µM). After these incubations, the cells were harvested on ice by mechanical scraping and lysed (0.5% SDS, 200 mM Tris-HCl pH 7.4, and 50 mM EDTA) on a glass slide. The slides were then tilted to allow DNA to spread along the slide. The samples were fixed in methanol–acetic acid (3:1) and washed with 70% ethanol. DNA was incubated with methanol for 5 min, washed with phosphate-buffered saline (PBS), denatured with 2.5 M HCl, and blocked with 5% bovine serum albumin (BSA; Sigma-Aldrich). IdU and CldU were stained at the same time using mouse anti-BrdU antibody (BD Biosciences, Franklin Lakes, NJ, USA) and rat anti-BrdU antibody (Bio-Rad Laboratories, Hercules, CA, USA), respectively, in PBS-T-BSA (0.05% Tween-20, 1% BSA). The slides were then washed with PBS-T followed by anti-mouse Alexa Fluor 594 and anti-rat Alexa Fluor 488 secondary antibodies (Thermo Fisher Scientific, Waltham, MA, USA). The slides were mounted using Fluoroshield (Sigma-Aldrich), and DNA fibers were imaged by a fluorescent microscope (Axiovert 200, Zeiss, Oberkochen, Germany) at a magnification of ×1000. Analyses were performed using the Zeiss LSM Browser Software. All experiments were performed at least twice, and a minimum of 150 fibers was counted for each slide.