Nbmpr

The following tritium radiolabeled compounds were used during the project: [³H]-Uracil (40 Ci/mmol), [³H]-Thymidine (71.7 Ci/mmol), [³H]-Uridine (60 Ci/mmol) were obtained from PerkinElmer (Waltham, MA, USA). [³H]-Cytidine (20 Ci/mmol) was purchased from American Radiolabeled Chemicals Incorporated (St Louis, MO, USA). Uridine, Uracil, Thymidine, 5-FlourUracil, 5-FlouroUridine, 2’-DeoxyUridine, 3’-deoxyThymidine, Sulfadiazine, NBMPR, Adenosine, Inosine, resazurin sodium salt and Phenyl Arsine Oxide were bought from Sigma-Aldrich (Poole, UK). 5-Flouro 2’DeoxyUridine was from VWR; 5-MethylUridine was from Alfa Aesar; 2’,3’-dideoxyUridine was from Carbosynth; Pyrimethamine was from Fluka; Ara-A was from ICN Biomedicals.

Treated cells were pre-incubated with various concentrations (from 1 μmol/L to 1 mmol/L) of dipyridamole (Dalian Mellon biological technology Co. Ltd, Dalian, China) and (from 1 nmol/L to 100 μmol/L) NBMPR (Sigma-Aldrich, St Louis, MO, USA), a hENT1 inhibitor, for 30 minutes before incubation with T₅₀-FAM or PMY_6-10-FAM for 30 minutes. The cells were then re-suspended for analysis by flow cytometry or confocal microscopy.

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.

Cells were treated with cytarabine (Selleck Chemicals, Houston, TX, USA), DNR (in-house), 6-thioguanine (Sigma Aldrich), or NBMPR (Sigma Aldrich). After 72–96 h, viable cells were counted on Vi-Cell Cell Viability Analyzer (Beckman Coulter, Krefeld, Germany). For long-term proliferation, cells were treated three times (d0, d4, d8) and viable cells were counted every second day. Unpaired, two-tailed Student’s t-test and calculation of IC₅₀ values were performed using GraphPad Prism version 6.07 (GraphPad Software, La Jolla, CA, USA). PiggyBac KDM6A cells were cultured +/− doxycycline (0.5 μg/mL) for 48 h followed by treatment with AraC +/− doxycycline for 72 h. For longer proliferation, cells were cultured +/− doxycycline (0.5 μg/mL) for 8 days. Every 2 days, cells were counted and cultured in fresh medium +/− doxycycline.
Additional methods are provided in supplementary methods.

Adenosine, ticagrelor, dipyridamole, NBMPR (S-(4-nitrobenzyl)-6-thioinosine), EDTA (ethylenediaminetetraacetic acid disodium salt dihydrate), 5-Iodotubericidin (4-Amino-5-iodo-7-(β-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidine), EHNA (erythro-9-amino-β-hexyl-α-methyl-9H-purine-9-ethanol hydrochloride) and PBS (phophate buffered saline, pH 7.4) were from Sigma-Aldrich. AOPCP (α, β-methyleneAdenosine 5′-diphosphate) was from Tocris. ¹³C₅-Adenosine, ¹³C₁₀-¹⁵N₅-Adenosine, ¹⁵N₅-AMP, ¹⁵N₅-Adenosine and ¹⁵N₄-hypoxanthine were from Cambridge Isotope Laboratories.