*4.4. Preparation of ABCG2 Variants Expression Vector*

To express human ABCG2 (NM\_004827.3) fused with EGFP at its N-terminus (EGFP-ABCG2) and EGFP (control), we used an ABCG2/pEGFP-C1 plasmid (open reading frame of ABCG2 was inserted into the *Hind*III and the *Apa* I sites of a pEGFP-C1 vector plasmid) that was from our previous study [32]. Of note, the functionality and expression of such construct were confirmed by previous studies we and other groups conducted [32,36,54–56]. Using a site-directed mutagenesis technique, the ABCG2 p.M131I (c.393G>T)/pEGFP-C1 plasmid and the ABCG2 R236X (c.706C>T)/pEGFP-C1 plasmid were generated from an ABCG2 WT/pEGFP-C1 plasmid, respectively. The introduction of each mutation was confirmed by full sequencing using BigDye Terminator v3.1 (Applied Biosystems, Foster City, CA, USA) and an Applied Biosystems 3130 Genetic Analyzer (Applied Biosystems), as described previously [40].
