SIRF assay

Cells were pulse treated with EdU, washed two times with PBS and subsequently treated with HU (0.2 μM) for 4 hr. Cells were fixed, permeabilized with 0.25% TritonX, and a click-iT reaction was performed using biotin azide (Life Technologies) according to manufacturer’s instructions. After incubation with primary antibodies, a Duolink proximity ligation assay (Sigma-Aldrich) was performed with mouse/rabbit detection red reagents according to the manufacturer's instructions. Slides were stained with DAPI and mounted with Prolong Gold before imaging using Nikon Eclipse Ti-U inverted microscope. Signals were analyzed using Duolink software, ImageJ and Nikon NIS elements, in addition to hand-counting of PLA signals. Data of repeated experiments were combined, and statistical analysis was performed using Prism6 software.