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1. Journal of Pharmacology and Experimental Therapeutics 163642 Potent and specific inhibition of mMate1-mediated efflux of type I organic cations in the liver and kidney by pyrimethamine Sumito Ito, Hiroyuki Kusuhara, Yushun Kuroiwa, Chunyong Wu, Yoshinori Moriyama, Katsuhisa Inoue, Tsunenori Kondo, Hiroaki Yuasa, Hideki Nakayama, Shigeru Horita, Yuichi Sugiyama (A) (B) (antibody) Anti - Myc b Anti - - actin mock mMATE1 - Myc (C) (D) Supplemental Figure 1. Construction of mouse Mate1 expressing HEK293 cells A) Western blot analysis of mMate1 in stably expressing HEK293 cells.Crude membrane fractions prepared from vector-transfected and mMate1-expressing cells were subjected to Western blot analysis. mMate1-Myc was detected with anti-c-Myc antibody. (B) Time profiles of cellular accumulation of [14C]TEA by mMate1. Vector-transfected (●) and mMate1-expressing HEK293 cells (○) were incubated with [14C]TEA (0.1  Ci/mL, 30 µM) at 37 ºC , and cellular accumulation was determined at designated time. (C) Concentration dependence of [14C]TEA uptake by mMate1. Vector-transfected (●) and mMate1-expressing HEK293 cells (○) were incubated with [14C]TEA (0.1 Ci/mL, 30 µM) and various concentrations of unlabled TEA at 37 ºC for 5 min, and cellular accumulation was determined at designated time. mMate1-mediated transport was calculated by subtracting the uptake in vector-transfected cells from that in mMate1-expressing cells. Concentration dependence of TEA uptake by mMate1 was shown by plotting uptake velocity against TEA concentration. (D) pH dependent uptake of TEA by mMate1. TEA uptake was measured in vector-transfected (●) and mMate1-expressing HEK293 cells (○) incubated for 5 min at the indicated pH. Each point represents the mean ± S.E. (n = 3) .

2. Journal of Pharmacology and Experimental Therapeutics 163642 Potent and specific inhibition of mMate1-mediated efflux of type I organic cations in the liver and kidney by pyrimethamine Sumito Ito, Hiroyuki Kusuhara, Yushun Kuroiwa, Chunyong Wu, Yoshinori Moriyama, Katsuhisa Inoue, Tsunenori Kondo, Hiroaki Yuasa, Hideki Nakayama, Shigeru Horita, Yuichi Sugiyama (A) (B) (C) (D) (E) (F) Supplemental Figure 2. Uptake of various compounds by mouse and human MATEs The uptake of various test compounds was measured by vector-transfected, mMate1, hMATE1, and hMATE2K expressing HEK293 cells. The cellular accumulation of test compounds was determined following a 5-min incubation at 37ºC. The concentrations of test compounds were (A) TEA (30 µM), (B) metformin (11 µM), (C) cimetidine (1 µM), (D) creatinine (1.2 µM), (E) carnitine (1 µM), (F) acyclovir (1 µM). Each point represents the mean ± S.E. (n = 3). *p<0.05, **p<0.01, ***p<0.001

3. Journal of Pharmacology and Experimental Therapeutics 163642 Potent and specific inhibition of mMate1-mediated efflux of type I organic cations in the liver and kidney by pyrimethamine Sumito Ito, Hiroyuki Kusuhara, Yushun Kuroiwa, Chunyong Wu, Yoshinori Moriyama, Katsuhisa Inoue, Tsunenori Kondo, Hiroaki Yuasa, Hideki Nakayama, Shigeru Horita, Yuichi Sugiyama