*4.5. Cell Culture and Transfection*

Human embryonic kidney 293 cell-derived 293A cells were purchased from Life Technologies (Carlsbad, CA, USA) and cultured in Dulbecco's Modified Eagle's Medium (DMEM; Nacalai Tesque, Kyoto, Japan) supplemented with 10% fetal bovine serum (Life Technologies), 1% penicillin/streptomycin, 2 mM L-glutamine (Nacalai Tesque), and 1 × Non-Essential Amino Acid (Life Technologies) at 37 ◦C in an atmosphere of 5% CO2. Each vector plasmid for ABCG2 WT, p.M131I, or p.R236X was transfected into 293A cells by using polyethyleneimine MAX (PEI-MAX; 1 mg/mL in milli-Q water, pH 7.0; Polysciences, Warrington, PA, USA) as described previously [57]. The amount of plasmid DNA used for transfection was adjusted per sample group.
