Native BrdU assay

Assessment of native BrdU levels by flow cytometry was performed as described [95]. Briefly, siRNA-transfected U-2 OS cells were grown in 20 μM BrdU-containing media for 48 h before being mock- or UV-treated. Cells were then harvested, washed once with cold PBS, and fixed with cold 100% EtOH at −20°C for at least 16 h. Cells were pelleted, washed and resuspended with PBS-T (PBS 1× + 0.1% Tween-20), and counted. Equal numbers of cells for each experimental condition were blocked with PBS-F (PBS 1× + 5% FBS) for 30 min at room temperature, washed with PBS-T, and then incubated with primary antibody (anti-BrdU; 1/100) in PBS-F buffer for 2 h at room temperature followed by incubation with Alexa Fluor–conjugated secondary antibody (1/200) in PBS-F for 1 h in the dark. Finally, cells were stained with DAPI and analyzed using an LSRII flow cytometer (BD Biosciences). The data were analyzed with FlowJo software (Flowjo LLC).