Uridine incorporation assay

Cells were treated as indicated. For the last 30 min of treatment, 200 μM 5-ethynyl-uridine (5-EU, ab146642, Abcam) was added to wells. For actinomycin D control samples, 4 µg/mL actinomycin D (A1410, Sigma-Aldrich) was added to wells 10 min prior to the addition of 5-EU. Cells were harvested by trypsinization and subjected to fixation/permeabilization in 125 mM PIPES (pH = 6.8), 10 mM EGTA, 1 mM MgCl_2, 3.7% formaldehyde and 0.2% Triton-X for 30 min at room temperature. Fixed and permeabilized cells were then stained using Click-iT Plus Alexa Fluor 647 Picolyl Azide Toolkit from Thermo Scientific ({"type":"entrez-nucleotide","attrs":{"text":"C10643","term_id":"1535714"}}C10643) according to the manufacturer’s instructions and analyzed by flow cytometry.