2.2. Establishment of Stable NTCP-Expressing Huh7.5 Cell Line (Huh7.5-NTCP)

Huh7.5 cells, a kind gift from Dr C. Rice (Rockefeller University, New York, NY, USA), were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma Aldrich, St. Louis, MO. D5796, high-glucose, with L-glutamine and sodium bicarbonate, without sodium pyruvate) supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin. Briefly, low-passage Huh7.5 cells were seeded at 4 × 10⁵ cells per well of a 6-well plate. To the lentiviral stock, polybrene was added to 4 μg/mL and HEPES (pH 7.0) to 20 mM. The lentivirus inoculum (1 mL) was added to each well intended for transduction. The 6-well plate was then centrifuged at 150× g for 1 h at 37 °C. Following centrifugation, the lentivirus was further incubated with the cells for 6 h at 37 °C in 5% CO₂. The medium for the transduced cells was then changed to DMEM containing 10% FBS. After 48 h of incubation at 37 °C, the medium was changed to DMEM containing 10% FBS and 0.1 μg/mL puromycin to select for cells that were successfully transduced. Transduced cells were cultured and selected in puromycin for one week prior to use in subsequent assays.

Overexpression of NTCP in transduced Huh7.5-NTCP cells was assessed and confirmed by RT-qPCR analysis of total RNA, flow cytometry analysis, and immunofluorescence staining of NTCP using the anti-NTCP antibody (Abcam, Cambridge, UK. ab175289). RT-qPCR was performed with the forward primer (5’-GGAGGGAACCTGTCCAATGTC-3′), reverse primer (5′-CATGCCAAGGGCACAGAAG-3′), and probe (5′-[6FAM]ACATGAACC/ZEN/TCAGCATTGTGATGACCACC-[IABk]-3′), all purchased from Integrated DNA Technologies (IDT, Coralville, IA). ΔΔCT values were calculated to determine fold change in NTCP mRNA expression. RT-qPCR for hypoxanthine-guanine phosphoribosyltransferase (HPRT) mRNA was performed using the Taqman primer probe mix from Applied Biosystems (Foster City, CA. cat No. 4326321E).