H 2000 10

Cells were seeded on fibronectin pre‐coated coverslips for 1 h, then washed with cold PBS, fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.1%Triton X‐100 for 5 min, blocked in blocking solution (3% bovine serum albumin and 10% goat serum in PBS) at room temperature for 1 h, and incubated with primary antibodies at 4°C in blocking solution overnight. On the second day, cells were washed with PBS and then incubated with secondary antibodies at room temperature for 1 h. Finally, cells were mounted onto slides with mounting medium (Vector Laboratory H‐2000‐10). Confocal images were obtained with a Leica confocal microscope and analysed with the FIJI Image J software.
The primary antibodies used were: Phalloidin (Thermofisher Scientific A‐21245 and A12381), Ki67 (Millipore AB9260), cleaved Caspase 3 (Cell Signalling Technology 9661S), Cyclin D1 (92G2 (Cell Signalling Technology 2978) and wheat germ agglutinin (WGA, Thermofisher Scientific W32466).

Cells plated on 96-well glass-bottom plates were incubated at 37°C and 5% CO₂ by an Okolab microscope stage incubator with 96-well insert during all imaging experiments. Confocal microscopy was performed on a spinning disk (Yokogawa CSU-X1) confocal microscope with an Andor DU-897 EMCCD camera on a Nikon Eclipse Ti body using a 100x oil immersion Apo TIRF objective (NA 1.49). The following wavelength lasers were used to image the respective constructs: constructs with mGFP (488 nm), mCherry (561 nm), miRFP (640 nm). Fixed samples in the 53BP1 counting assay also used the 405 nm laser to detect nuclei stained with Hoechst (Thermo Fisher Scientific, H3570) or DAPI (Vectashield, H-2000-10).