14c choline

The RNA was extracted by using the E.Z.N.A.® Total RNA Kit I (Omega Bio-tek, Norcross, GA, USA) and converted into cDNA by using M-MuLV reverse transcriptase (400 U), random hexamers (20 µg) and dNTPs (10 mM) (Fermentas, Hanover, MD, USA). Quantification of the MATE1-2 and PMAT genes was performed by employing the Prism 7500 sequence detection system (Applied Biosystems, Inc., Foster City, CA, USA). Briefly, 6 µL of each sample was mixed with 10 µL of PCR reagent mixture (0.5 µL primer probe mix, 5 µL of TaqMan master mix (TaqMan Gene Expression assay, Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA) and 0.5 µ sterile water). Transporter gene expression was determined by real-time polymerase chain reaction (RT-PCR) and normalized to endogenous cyclophilin A. The used primer probe mixes included HS00928283 (PMAT and SLC9A4), Hs00217320_m1 (MATE1 and SLC47A1) and Hs00945652_m1 (MATE2 and SLC47A2).
OCT transporters in MCF-7 and MDA-MB-231 cell lines were characterized previously [13 (link)]. The studies included characterization of the concentration dependency of choline chloride uptake and time dependency of [¹⁴C] choline (PerkinElmer, Waltham, MA, USA) uptake. Furthermore, the function of OCT transporters (OCT 1–3) was checked using three known OCT inhibitors: disopyramide, naringin and cimetidine [13 (link)].