Net355001mc

[³H]-thymidine (Perkin Elmer NET355001MC, PerkinElmer, Waltham, MA) was utilized for in vitro assessment of therapy-induced changes in thymidine salvage pathway activity in cultured human NSCLC cells. [³H]-thymidine specific activity was >10Ci(370GBq)/mmol and radiochemical purity >97%. H460 and H1299 cells were seeded (1×10⁶/well) in 6-well plate in RPMI1640 supplemented with 10% FBS and antibiotics, incubated 24 hours in 5% CO₂ at 37°C. When cell cultures achieved 80% confluence, cells were exposed to treatment with either the vehicle (sterile water), pemetrexed (100 nM), or the combination of pemetrexed (100 nM) and cisplatin (10 μM) in growth media for varying exposure times ranging up to 48 hours. Drug-containing medium was then removed, and the cells were then washed and pulsed with 5 μCi [³H]-thymidine/well for 1 hour. The cells were then washed and scraped into plastic vials. Scintillant (10 mL; Research Products International Corp., Mount Prospect, IL) was added to each vial and the radioactivity was counted on a scintillation counter (Beckman Coulter LS6500, Beckman Coulter Life Sciences, Indianapolis, IN).

Details of the thymidine proliferation assay have been previously described^{44 (link),53 (link)}. Briefly, PBMCs from HBV donors were isolated and were cultured with RPMI containing 2% human AB serum with pen/strep for 14 days in the presence of medium, HBV overlapping peptides of S or Core, influenza overlapping peptides, as well as small molecules (0.5 μM), or antibodies (5 μg/μl), including atezolizumab and nivolumab^{44 (link)}. HBV genotype-specific overlapping 15-mer peptides (genotypes A–D) were synthesized by GenScript (NJ, USA). A total of 1 μCi of ³H thymidine (NET355001MC, PerkinElmer) was added for 18 h for its incorporation into newly synthesized chromosomal DNA before harvesting cells. ³H thymidine incorporation was counted by MicroBeta2 microplate scintillation counter (PerkinElmer, MA). A total of 50 μl of supernatants per well before adding ³H thymidine was used for measuring IFNγ production by ELISA (430107, BioLegend).

[³H]-thymidine (Perkin Elmer NET355001MC, PerkinElmer, Waltham, MA) was utilized for in vitro assessment of therapy-induced changes in thymidine salvage pathway activity in cultured human NSCLC cells. [³H]-thymidine specific activity was >10Ci(370GBq)/mmol and radiochemical purity >97%. H460 and H1299 cells were seeded (1×10⁶/well) in 6-well plate in RPMI1640 supplemented with 10% FBS and antibiotics, incubated 24 hours in 5% CO₂ at 37°C. When cell cultures achieved 80% confluence, cells were exposed to treatment with either the vehicle (sterile water), pemetrexed (100 nM), or the combination of pemetrexed (100 nM) and cisplatin (10 μM) in growth media for varying exposure times ranging up to 48 hours. Drug-containing medium was then removed, and the cells were then washed and pulsed with 5 μCi [³H]-thymidine/well for 1 h. The cells were then washed and scraped into plastic vials. Scintillant (10 ml; Research Products International Corp., Mount Prospect, IL) was added to each vial and the radioactivity was counted on a scintillation counter (Beckman Coulter LS6500, Beckman Coulter Life Sciences, Indianapolis, IN). For ENT1 inhibition, cells were incubated with NBMPR (Nitrobenzylthioinosine) for 15min (100uM; Sigma-Aldrich, St. Louis, MO) prior to labeling with [³H]-thymidine.