3h uracil

[¹⁴C]Urate (55.0 mCi/mmol), [³H]guanine (10.7 Ci/mmol), [³H]hypoxanthine (27.0 Ci/mmol), [³H]thymine (65.0 Ci/mmol), and [³H]uracil (42.8 Ci/mmol) were obtained from Moravek Biochemicals (Brea, CA), [¹⁴C]inulin (1.9 mCi/g) was from American Radiolabeled Chemicals (St. Louis, MO), and [³H]polyethylene glycol 4000 (PEG 4000, 1.5 mCi/g) was from PerkinElmer Life Sciences (Boston, MA). Unlabeled urate, guanine, hypoxanthine, thymine, and uracil were obtained from Wako Pure Chemical Industries (Osaka, Japan), and Ko143 was from Sigma‐Aldrich (St. Louis, MO). Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were obtained from Wako Pure Chemical Industries and Invitrogen (Carlsbad, CA), respectively. Mouse monoclonal antibodies for the tag peptides of DYKDDDDK (FLAG) and hemagglutinin (HA) were obtained from Wako Pure Chemical Industries (product numbers of 014‐21881 and 018‐22381, respectively, for the anti‐FLAG and anti‐HA antibodies), and a mouse monoclonal antibody for β‐actin (product number A5441) and horseradish peroxidase‐conjugated goat anti‐mouse IgG (product number A8924) were from Sigma‐Aldrich. All other reagents were of analytical grade and commercially obtained.

[³H]Adenine (25.0 Ci/mmol), [³H]hypoxanthine (27.0 Ci/mmol), [³H]thymidine (20.0 Ci/mmol), [³H]uridine (14.7 Ci/mmol), [³H]uracil (42.8 Ci/mmol), [³H]xanthine (12.8 Ci/mmol) and [³H]adenosine (39.2 Ci/mmol) were obtained from Moravek Biochemicals (Brea, CA), [¹⁴C]guanine (55 mCi/mmol) was from American Radiolabeled Chemicals (St. Louis, MO), and [¹⁴C]ascorbate (8.5 mCi/mmol) was from PerkinElmer Life Sciences (Boston, MA). Adenine, papaverine, and dipyridamole were obtained from Sigma-Aldrich (St. Louis, MO), and hypoxanthine, guanine and nitrobenzylthioinosine were from Wako Pure Chemicals (Osaka, Japan). All other reagents were of analytical grade and commercially obtained.

VERO, (2 × 10⁴ cells/100 µl/well) were grown in complete medium (IMDM) on 96-well plates. After 24 h incubation, a medium was removed and then T. gondii RH tachyzoites, suspended in culture medium supplemented with 0.9–125.00 μg/ml diaminotriazines 4a–l and as a control-sulfadiazine (5.0–2500.0 μg/ml), were added (2 × 10⁵ tachyzoites/200 µl/well) to the cell monolayers. After subsequent 48 h incubation 1 µCi/well of [³H] uracil (Moravek Biochemicals Inc., Brea, CA, USA) was applied to each microculture for further 18–20 h. The amount of the isotope incorporated into the parasite nucleic acid pool, corresponding to the parasite growth, was measured by liquid scintillation counting with 1450 Microbeta Plus Liquid Scintillation Counter (Wallac Oy, Turku, Finland). The cpms of host cells alone (below 250/microculture) were subtracted from cpms of T. gondii infected microcultures (Dzitko et al. 2014 (link)).