Metacyte

To control for the non-dividing status of the confluent cell layer, cells were evaluated by eye and routinely tested by cell cycle analysis. For this, resting and exponentially growing cells were incubated for 3 h with 5 µM of the nucleotide analogue EdU. Cells were fixed using 3% para-formaldehyde and permeabilized with 0.2% Triton in PBS for 10 min at RT. EdU incorporated during the S phase of the cell cycle was stained via a click-it reaction according to the manufacturer’s instructions (PANATecs). DNA was stained with DAPI (0.2 µg/ml) for 5 min. Cells were washed again with ultrapure water, mounted with VECTASHIELD mounting medium (Vector Laboratories) and sealed with nail polish. The population was automatically scanned at a widefield microscope (Axiovert 200 M with a 20x Plan-Apochromat objective, Zeiss) and the EdU signal intensity was plotted vs. the DAPI signal intensity by using the program MetaCyte (MetaSystems). Non-dividing G0/G1 cells have a low EdU and a low DAPI signal (Fig. S1a,b).

To determine repair kinetics of radiation-induced DSBs we used a γH2AX-foci analysis according to Löbrich, et al. [45 (link)]. γH2AX foci were quantified at different time points after irradiation. γH2AX-foci numbers of non-irradiated cells were subtracted and the data were normalized to data 15 min after irradiation, which were considered 100% DSB damage upon irradiation. Thus, the resulting ratio represents the fraction of remaining DSBs after irradiation. The absolute number of γH2AX foci 15 min after irradiation was similar between the tested genotypes. The cell-cycle phase of each cell was determined based on its nuclear DAPI signal with the software MetaCyte (MetaSystems, Heidelberg, Germany). Adding EdU after irradiation allowed distinguishing G2 cells that were irradiated in G2 phase from G2 cells that were irradiated in a previous cell-cycle phase. Only G2 cells that were irradiated in G2 phase were analyzed. Using Metafer 4 (MetaSystems), images of the DNA, EdU, and γH2AX signal were taken and analyzed automatically resulting in DSB-repair kinetics. Per time point and cell-cycle phase, approximately 300 cells on average were analyzed.