Functional Assay of Transporters in HEK293 Cells.

HEK293 cells were seeded in poly-D-lysine coated 24-well plates and transfected with 500 ng of cDNA per well using Lipofectamine 2000 or Fugene HD (for the kinetics). Transport assays were performed 24 h or for the kinetics 48 h post-transfection. The amount of E3S transported into cells was measured by liquid scintillation counting. Initial experiments showed that OATP1B3-mediated uptake of 0.1 μM E3S was linear up to at least 1.5 min. Therefore, uptake and kinetic experiments for E3S were performed at 1 min, and its procedure was the same as described in our previous publication.⁸ In all experiments, cells transfected with empty vector were used as background control. Transporter-specific uptake was calculated by subtracting the background uptake from OATP-transfected uptake and normalized with total protein concentration which was determined by the BCA assay.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was employed to quantify the amount of EGCG transported into cells. Chromatographic separation was achieved with an Agela Venusil C₁₈ column (2.1 mm × 50 mm, 5 μm) and the flow rate was set at 0.4 mL/min. The mobile phase consisted of water containing 0.1% acetic acid (A) and acetonitrile containing 0.1% acetic acid (B) with the following gradient: 0–0.5 min, 3% B; 2 min, 28% B; 2.0–8.0 min, 28% B; and 8.1–10.0 min, 3% B. The mass spectrometer was operated in negative electrospray ionization (ESI) mode and quantitation was performed by multiple reaction monitoring (MRM). The ion transitions for EGCG and internal standard (IS) epicatechin gallate (ECG) were selected as m/z 457.3→169.0 and m/z 440.7→168.8, respectively.