2.3. Cellular uptake experiment

The cellular accumulation of ETV in hepatic cell lines or PHH was performed as described in our previous study (Li et al., 2016). Briefly, the cells were pre‐incubated with HBSS at 37°C for 10 min with or without inhibitors, and then HBSS containing ETV in the absence or presence of inhibitors was added to initiate the uptake. The uptake was terminated by removing the incubation buffer and adding ice‐cold PBS quickly at the designated time. Then the cells were washed three times with ice‐cold PBS and lysed with 100 μl of 0.1% sodium dodecyl sulfate.

The intracellular accumulation of ETV, ribavirin, and cGMP was also determined in OAT2, ENT1 transfected, or mock cells using the method described for hepatic cell lines, except the buffer was replaced by Na⁺‐free KRH (NMDG, 125 mM; d‐glucose, 5.6 mM; KCl, 4.8 mM; MgSO₄·7H₂O, 1.2 mM; KH₂PO₄, 1.2 mM; CaCl₂, 1.2 mM; HEPES, 25 mM; pH = 7.4) for hENT1, or HBSS containing 10 μM of NBTI for hOAT2, respectively.

The concentrations of ETV, cGMP, and ribavirin in the cells were quantified with LC–MS/MS and normalized to the total protein content, detected by use of a BCA assay, in the lysates to control for variations in cell numbers. The uptake in the presence of inhibitors was expressed as a percentage of the vehicle group (% of control).