Overexpression of ABCG2 and URAT1 in 293A Cells

Human embryonic kidney 293 cell-derived 293A cells were purchased from Life Technologies (Carlsbad, CA, USA) and cultured in DMEM (Nacalai Tesque) supplemented with 10% fetal bovine serum (Biowest, Nuaillé, France), 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 as described previously (Toyoda et al., 2016a (link)). All experiments were carried out with 293A cells at passages 10–20. To express human ABCG2 (NM_004827) fused with Myc-tag at its N-terminus (Myc-ABCG2) and EGFP (control), we used Myc-ABCG2 and EGFP-expressing adenoviruses constructed in our previous study (Ito et al., 2015 (link)), respectively. To express the URAT1, open reading frame of URAT1 (NM_144585.3) was cloned into a pcDNA3.1(+) vector (Life Technologies) with a FLAG tag at its N-terminus. To express mouse Abcg2 and EGFP (control), open reading frames of mouse Abcg2 (NM_011920) and EGFP were inserted into a pcDNA3.3 vector (Life Technologies), respectively.