DNA fiber analyses were carried out as reported with minor modification (33 (link)). Briefly, cells were first pulse-labeled with 25 μM IdU for 30 min, washed three times with PBS, then pulse-labeled with 250 μM CldU for 30 min followed by 4 hrs of treatment with 2mM HU. Cells were harvested and resuspended in PBS for a final concentration of 1500 cells/μl. Cells were then lysed in lysis buffer (200 mM Tris–HCl (pH 7.5), 50 mM EDTA, 0.5% SDS), DNA fibers were stretched onto positively charged glass slides and fixed in methanol:acetic acid (3:1). After rehydration in PBS twice for 5 min, DNA were denatured with 2.5 M HCl for 1 h, slides were then washed with PBS for three times and blocked with 5% BSA at 37°C for 1 h. The newly replicated ldU and CIdU tracks were immunostained using mouse-anti-BrdU (1/100, 347580, BD Biosciences) and rat-anti-BrdU (1/100, Ab6326, Abcam) primary antibodies, and Alexa Fluor 546 goat-anti-mouse IgG1 (1/500, A-21123, ThermoFisher Scientific) and Alexa Fluor 488 goat-anti-rat (1/500, A-11006, ThermoFisher Scientific) secondary antibodies respectively. Coverslips were mounted using ProLong Gold Antifade Mountant (ThermoFisher Scientific).