Profiling Homologous Recombination Efficiency

For proficiency analysis of homologous recombination, cells were seeded on cover slides to 90% confluence [14 (link)]. The cells were treated with CC-115 (5 μM; CAS No. 1228013-15-7) and subsequently irradiated (10 Gy dose) 24 h afterwards. The cells were fixed and permeabilized after 4 h (4% formaldehyde and 0.1% Triton ×−100/PBS). After blocking (1% bovine serum albumin; SERVA Electrophoresis GmbH, Heidelberg, Germany), the slides were stained with the primary antibodies mouse anti-γH2AX (1:1500, Merck, Darmstadt, Germany) and rabbit anti-Rad51 (1:250, abcam, Cambridge, UK) and with the secondary antibodies AlexaFluor488 goat anti-mouse and AlexaFluor594 chicken anti-rabbit (Invitrogen, Eugene, OR, USA). The nuclei were stained using DAPI (Sigma Aldrich, St. Louis, MO, USA). The image acquisition was performed by a Zeiss Axio Plan 2 fluorescence microscope (Zeiss, Göttingen, Germany) and an automated quantification was done using Biomass Software (MSAB, Erlangen, Germany).