Immunofluorescence

For immunofluorescence, cells were fixed with 3.7% formaldehyde/1× PBS (Cat.No.: F8775, Sigma-Aldrich Chemie GmbH, Steinheim, Germany) for 15 min and permeabilized with 0.7% Triton-X100 in 1× PBS for 20 min. All washing steps were performed with PBS-T (1× PBS/0.075% Tween-20). For detection of PCNA, cells were further incubated for 5 min in ice-cold methanol for antigen retrieval. Blocking (1% bovine serum albumin in 1× PBS) was performed for 30 min at room temperature. EdU was detected using the Click-IT assay as described by the manufacturer (1:1000 6-FAM azide or 1:2000 5/6-sulforhodamine azide; Cat.No.: 7806 and 7776, respectively, Carl Roth, Karlsruhe, Germany). Primary and secondary antibodies were diluted in the blocking buffer and incubated for 1 hr at room temperature with subsequent 3×10 min of PBS-T washing. DNA was counterstained with DAPI (4′,6-diamidino-2-phenylindole, 10 μg/ml, Cat.No.: {"type":"entrez-nucleotide","attrs":{"text":"D27802","term_id":"522535","term_text":"D27802"}}D27802, Sigma-Aldrich Chemie GmbH, Steinheim, Germany) for 10 min, and samples were mounted in Vectashield (Cat.No.: VEC-H-1000, Vector Laboratories, Inc, Burlingame, CA, USA). Antibody characteristics are summarized in Supplementary file 1d.