Live-cell and Confocal Microscopy Imaging

Live-cell microscopy was performed using a BD pathway 855 High-Content Bioimager system (BD Biosciences, Franklin Lakes, NJ) with a 20× objective (numerical aperture [NA], 0.75) at a humidified 37°C with 5% CO₂. Images were taken using Attovision software (BD Biosciences) and analyzed using ImageJ as described previously (Ma et al., 2012 (link)). Duration of G1 phase was determined by counting frames between chromosome segregation and the appearance of PCNA-YFP punctum imaged by fluorescence microscopy. Confocal microscopy was performed during DNA fiber analysis on a Nikon A1 scanning confocal microscope (Nikon, Tokyo, Japan) equipped with 561- and 633-nm lasers. Images were taken acquired by 60× oil-immersion objective (NA 1.2) and an Orca-Flash 4.0 sCMOS camera (Hamamatsu, Hamamatsu, Japan) controlled by Nikon Element software. Fixed-cell microscopy for immunofluorescence was performed using a Leica DM5500B (Leica, Wetzlar, Germany) with a 20× objective and a Retiga Exi FAST1384 charge-coupled device camera (Qimaging, Surrey, Canada). Camera and filters were controlled via Image-Pro (Media Cybernetics, Bethesda, MD).