*4.7. Immunofluorescence*

Cells were collected by trypsinization and 5 × 10<sup>4</sup> cells were replated onto coverslips. The cells were fixed with cold methanol for 10 min on ice followed by washing with 1× PBS<sup>−</sup>. Then, the primary antibodies diluted in TBS-DT (20 mM Tris-HCl, pH 7.6, 137 mM NaCl, 0.1% Tween 20, 125 μg/mL ampicillin, 5% skim milk) were treated for 2 h at 37 ◦C, followed by the Alexa Fluor-labeled secondary antibodies for 1 h at 37 ◦C. Nuclei were counterstained with 1 μg/mL DAPI. The antibodies used was anti-53BP1 (A300-272A, BioLegend, San Diego, CA, USA), and Alexa Fluor 555-labed anti-rabbit IgG (A21428, Thermo Fisher Scientific, Waltham, MA, USA). Images were captured by fluorescence microscope (DM6000B, Leica, Tokyo, Japan) and analyzed by FW4000 (Leica, Tokyo, Japan).
