Cells were plated at ~30% confluency and treated with 0, 200, or 400 nM palbociclib for 24 hours. During the final 45 min of treatment, culture medium was supplemented with 10 μM 5-bromo-2′-deoxyuridine (BrdU). At the completion of treatment, cells were trypsinized. Once the cells had detached from the tissue culture plate, the trypsin was neutralized with complete media and the cells were counted using a Vi-CELL XR (Beckman Coulter). One million cells were pelleted, and the supernatant was aspirated. Cells were then washed once with 1× PBS and pelleted. Cells were fixed, stained, and analyzed by flow cytometry using the FITC BrdU Flow Kit (BD Pharmingen, no. BD559619) according to the manufacturer’s instructions. Histograms were created using FlowJo.

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