FIG 1.

Characterization of mouse and human cell lines stably expressing hNTCP. AML12 (obtained from ATCC CRL-2254), mH274#26 (8), and HepG2 cells were transduced with lentiviruses coexpressing hNTCP and a puromycin resistance gene and selected to establish a stable cell pool. (A) Total cell lysates of the transduced (+) or parental (−) cells were treated with PNGase F (New England BioLabs) and analyzed by Western blotting using a hNTCP-specific antibody (Sigma-Aldrich). (B) The respective transduced (lower row) and parental (upper row) cell lines were incubated for 30 min with 200 µM atto565-labeled Myrcludex B, washed, and analyzed by fluorescence microscopy. (C) Cell lines were incubated with ³H-TC (Hartmann Analytic) for 15 min at 37°C, washed, and lysed, and scintillation counts of the total lysates were measured and the results normalized to the cell number. TC uptake was inhibited by treatment of the cells with 2 μM MyrB (13).