Ab1837

Cells were seeded onto either 96-well glass-bottom plates and grown overnight. Cells treated with zeocin for specified time and concentration were fixed with 4% formaldehyde. Then, 0.1% Triton-X was used to permeabilize the cells, followed by blocking with 3% bovine serum albumin. Cells were then incubated with the following antibodies: phospho DNA-PKcs S2056 (ab18192, Abcam, 1:200), RAD51 (ab1837, Abcam, 1:200), RPA70 (ab176467, Abcam, 1:200), cyclin E (4132S, Cell Signalling), or HA-tag (#3724, Cell Signalling, 1:100). The cells were washed and stained with the appropriate secondary antibodies: Alexa Fluor 594 (red) goat anti-rabbit (A11012, Thermo Scientific), Alexa Fluor 488 (green) goat anti-mouse (A11001, Thermo Scientific). After washing, the cells were stained with 10 μM DAPI in PBS and visualized with the Zeiss LSM 770 microscope. Images were analyzed using the ImageJ techniques previously described [72 (link)]. Cyclin E intensity was measured for each cell. Average cyclin E intensity of cells grown in media without growth factor for 4 hours was used to define the threshold of cyclin E positive. Colocalized foci appear yellow when green and red channels are merged in ImageJ.

Cells were collected from 6 cm plates, at about 80–90% confluence, by using trypsinization. Cells were washed with cold PBS and fixed with 95% cold ethanol for 10 min at –20°C. Cells were stained with anti-RAD51 antibody (ab1837, Abcam, 1:100) and Alexa Fluor 488 goat anti-mouse (A11001, Thermo Fisher Scientific). After washing, cells were resuspended in 200 µL PBS and NUCLEAR-ID Red DNA stain (ENZ-52406, Enzo Life Science), and incubated in the dark at room temperature for 30 min. Samples were analyzed by a BD Accuri C6 Plus Flow Cytometer.

Cells were seeded onto either 96-well glass-bottom plates and grown overnight. Cells treated with zeocin (10 μg/mL, 10 min) were fixed with 4% paraformaldehyde. Then, 0.1% Triton-X was used to permeabilize the cells, followed by blocking with 3% bovine serum albumin. Cells were then incubated with the following antibodies: RAD51 (ab1837, Abcam, 1:200), cyclin E (4132S, Cell Signaling), pH2AX S139 (9718S, Cell Signaling), and cyclin A (ab39, Abcam). The cells were washed and stained with the appropriate secondary antibodies: Alexa Fluor 594 (red) goat anti-rabbit (A11012, Thermo Fisher Scientific), Alexa Fluor 488 (green) goat anti-mouse (A11001, Thermo Fisher Scientific). After washing, the cells were stained with 10 µM DAPI in PBS and visualized with the Zeiss LSM 770 microscope. Images were analyzed using the ImageJ techniques previously described (Murthy et al., 2018 (link)). cyclin E intensity was measured for each cell. Average cyclin E intensity of cells grown in media without growth factor for 4 hr was used to define the threshold of cyclin E positive.