53BP1 immunofluorescence cellular assay was performed as previously described (Taglialatela et al., 2021 (link)). SUV420 KO, SUV420H1, SUV420H1R286A and SUV420h1ΔC-terminus mouse fibroblast cells were collected, counted, and 6000 cells were seeded on black 96-well bottom-glass plates. After 24h the cells were exposed to 1 μM etoposide for 30 min and given 24h, 4h, 2h, 1h or 0h to recover after etoposide removal before the cells were simultaneously fixed and permeabilized (4% paraformaldehyde, 0.5% Triton X-100) for 10 min at room temperature. Cells were incubated in blocking solution (3% BSA in TBS-Tween 0.1%) for 1h with primary antibody (anti-53BP1, Rabbit polyclonal, Bethyl #A300–272A) diluted in blocking solution overnight at 4°C. After three washes with TBS-T the cells were incubated for 1 hr at room temperature with the secondary antibody Alexa Fluor 488-labeled anti-rabbit at 1:1,000 dilution (A-11008, Thermo Fisher Scientific). After three washes in TBS-T, cells were incubated with DAPI for 5 min at room temperature to stain nuclei. Image acquisitions were made using the ImageXpress Nano Automated Imaging System microscope (Molecular Devices).The imaging software MetaXpress 6 was used to automate image analysis. The experiment was done with three biological replicates for each clone.