Cellular Uptake of Azacitidine Kinetics

IUR [40 (link)] were performed in triplicate with 2 × 10⁵ cells per tube. Briefly, 2 × 10⁵ cells were incubated for 2 h at 37 °C in the presence and absence of a 50% ¹⁴C-labeled 2 μM AZA. For IUR with inhibitors, 100 μM verapamil (Royal Adelaide Hospital Pharmacy, Adelaide, SA, Australia), 200 μM procainamide (Sigma-Aldrich, St. Louis, MO, USA), 10 μM corticosterone (Sigma-Aldrich, St. Louis, MO, USA), 20 μM NBMPR (Sigma-Aldrich, St. Louis, MO, USA), 20 μM cyclosporin A (Sigma-Aldrich, St. Louis, MO, USA), 10 μM chloroquine (Sigma-Aldrich, St. Louis, MO, USA), 150 μM amantadine (Sigma-Aldrich, St. Louis, MO, USA), 20 and 200 μM cimetidine (Sigma-Aldrich, St. Louis, MO, USA), and 0.1 and 10 μM pyrimethamine (Sigma-Aldrich, St. Louis, MO, USA) were added. After incubation the cellular and aqueous phases were separated, and incorporation determined using a Perkin Elmer Liquid Scintillation Analyser following the addition of Microscint 20 scintillation fluid (Perkin Elmer, Waltham, MA, USA) before counts per minute of β radiation in the supernatant and cell pellet fractions was used to convert to ng of AZA in 2 × 10⁵ cells. All assays were performed in triplicate and repeated if the assay demonstrated non-concordance.