Ketoconazole

CEL (purity ≥98%), ketoconazole (as internal standard [IS]; purity ≥98%), as shown in Figure 1, and REP (purity >98%) were purchased from Tokyo Chemical Industry Co. (Tokyo, Japan). DMSO, ethanol, potassium phosphate monobasic/dibasic, and polyethylene glycol 400 were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). β-nicotinamide adenine dinucleotide phosphate (NADPH), HLM, and RLM were purchased from BD-Genetech (Woburn, MA, USA). ACN and methanol of HPLC grade were purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA).

Yeast media were prepared as described in Adams et al. (1997) . Gene disruptions were performed as detailed in Sikorski and Hieter (1989) (link) and Taxis and Knop (2006) (link). Strains were cultured at 30° except where otherwise indicated. The genotypes of strains used in the course of this study, along with construction details, are provided in Supporting Information, Table S1. Oligonucleotides used during this study are listed in Table S2. Ethidium bromide (Thermo-Fisher Scientific, Waltham, MA) was used at a concentration of 25 µg/mL to destroy mtDNA. Cycloheximide (CHX; Sigma-Aldrich, St. Louis, MO) was used at a concentration of 10 µg/mL for plasmid counterselection on yeast extract peptone dextrose broth (YEPD) medium, 3 µg/mL for plasmid counterselection on yeast extract peptone 3% glycerol+3% ethanol (YEPGE) medium, and at 0.2 µg/mL in YEPD medium to test for activation of the PDR pathway. Ketoconazole (Tokyo Chemical Industry Co., Tokyo, Japan), another PDR substrate, was used at a concentration of 2 µg/mL in YEPD. To disrupt the actin cytoskeleton in logarithmic-phase cultures, latrunculin A (Santa Cruz Biotechnology, Dallas, TX) was used at a concentration of 10 µM in SD medium lacking leucine (SD-Leu), following the procedure of Hammermeister et al. (2010) (link). Serial dilution assays were performed as in Garipler et al. (2014) (link).

Acacetin (purity > 98%) and ketoconazole (KCZ) were purchased from Tokyo Chemical Industry Co. (Tokyo, Japan). Chlorpropamide (purity > 97%), quinidine (QND), sulfaphenazole (SPZ), α-naphthoflavone (NF), phosphate-buffered saline (PBS), dimethyl sulfoxide (DMSO), and polyethylene glycol 400 (PEG 400) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Nicotinamide adenine dinucleotide phosphate (NADPH), uridine diphosphate glucuronic acid (UDPGA), and rat liver microsomes (RLM; from male Sprague-Dawley rats) were purchased from Corning, Inc. (Midland, NC, USA). Pooled plasma (from male Sprague-Dawley rats) was purchased from Innovative Research, Inc. (Novi, MI, USA). The protocol for the animal studies conducted in this report was approved by the Institutional Animal Care and Use Committee at Pusan National University (approval no. PNU-2019-2217, date of approval: 17 April 2019). Eight-week-old male Sprague-Dawley rats weighing approximately 250 g were purchased from DBL Co. (Chungcheongbuk-do, Korea). The rats were housed and acclimatized before use, as reported previously [22 (link)].