Rifampicin

All the chemicals and reagents used in this study were of analytical grade. GPI0363 was obtained from the chemical library of the Drug Discovery Initiative at the University of Tokyo and its purity was confirmed by ultra-performance liquid chromatography-high resolution mass spectrometry (m/z: 343.1968 [M + H]⁺; calculated for C₁₉H₂₄N₄O: 343.1934) (ESI Fig. 7†). Rifampicin (potency: 1029 μg mg⁻¹), ammonium acetate, and HPLC grade acetonitrile were obtained from Fujifilm Wako Pure Chemical Industries, Ltd., Osaka, Japan. Fidaxomicin (purity: ≥95%) was obtained from Cayman Chemical (Ann Arbor, MI, USA). Salmon sperm DNA, actinomycin D (purity: ∼98%), and phenol : chloroform : isoamyl alcohol (125 : 24 : 1, v/v/v) were obtained from Sigma Aldrich, Tokyo, Japan. Tryptone, tryptic soy broth (TSB), yeast extract, and Mueller-Hinton broth (MHB) were obtained from Becton, Dickinson and Company (Franklin Lakes, NJ, USA). RNase inhibitor was obtained from Applied Biosystems (Beverly, MA, USA). ATP, CTP, GTP, UTP, and yeast tRNA were obtained from Ambion (Austin, TX, USA). [α-³²P] UTP was obtained from PerkinElmer, Waltham, MA, USA. The E. coli RNAP holoenzyme and core enzyme were obtained from New England Biolabs (Ipswich, MA, USA), and TALON® magnetic beads were obtained from Takara Bio USA Inc. (Mountain View, CA, USA).

Cells cultured in the 24-well plates in ESTEM-HE medium were treated with 100 nM vitaminD3 (D1530-10UG, Sigma-Aldrich)or 40 μM β-naphthoflavone (N3633-1G, Sigma-Aldrich) or 50 μM omeprazole (150–02091, Fujifilm Wako Pure Chemicals Co., Ltd.) for 24 h or 20 μM rifampicin (189–01001, Fujifilm Wako Pure Chemicals Co., Ltd.) or 500 μM phenobarbital(162–11,602, Fujifilm Wako Pure Chemicals Co., Ltd.) or 100 μM dexamethasone (194,561, MP Biomedicals, Illkirch, France) for 48 h at a cell density of 80%. Controls were treated with DMSO (final concentration 0.1%).

To examine the induction of cytochrome P450 (CYP) enzymes, cells were cultured in 12-well plates (353043, BD Falcon; Corning, NY, USA) using irrMEF and EMUKK-05 medium. When the cells reached 80–90% confluence, the following drugs were added to the cells: 20 μM rifampicin (solvent: DMSO, 189–01001, Fujifilm Wako Pure Chemicals, Osaka, Japan) with an induction period of 2 days, 50 μM omeprazole (solvent: DMSO, 158–03491, Fujifilm Wako Pure Chemicals, Osaka, Japan) with an induction period of 1 day and 500 μM phenobarbital (solvent: DMSO, 162–11602, Fujifilm Wako Pure Chemicals, Osaka, Japan) with an induction period of 2 days.

Transient gene expression in protoplasts was performed using the polyethylene glycol (PEG)-calcium transfection method^{69 (link)}.
For Agrobacterium infiltration, individual pRI101 plasmids were transformed into Agrobacterium tumefaciens GV3101. Transient gene expression in N. benthamiana leaves was performed using the Agrobacterium infiltration method originally developed by Goodin et al.^{70 (link)}. Transformed Agrobacterium cells were cultured in LB medium containing 50 µg/ml rifampicin (Wako, Osaka, Japan), 20 µg/ml gentamicin (Wako) and 50 µg/ml kanamycin (Wako) at 28 °C for 24–36 hr. Cells were harvested and washed with MES buffer (10 mM MES (pH 5.6), 10 mM MgCl₂, 100 µM acetosyringone) and adjusted to an OD₆₀₀ of 1.0 with MES buffer. For co-infiltration of Agrobacterium carrying different plasmids such as GFP and TagRFP, the mixing volume ratio between GFP and TagRFP was 1:2. After incubation for 2–3 hr at room temperature, the Agrobacterium suspension was slowly infiltrated into the leaf abaxial surface using a 1 ml syringe. After incubation for 40–48 hr in a growth chamber, the infiltrated leaves were observed.

Camptothecin and SN-38 were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Ko143 was purchased from Sigma Aldrich Co. LLC (St. Louis, MO, USA). Verapamil and rifampicin were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Baicalin was purchased from AdooQ Bioscience (Irvine, CA, USA), and Cell Quanti-Blue^TM Cell Viability Assay Kit was purchased from Bio Assay Systems (Hayward, CA, USA). All other reagents were of high-purity analytical or high-performance liquid chromatography (HPLC)-grade. Pooled human serum from healthy subjects (normal serum) was purchased from Merck Millipore Ltd. (Billerica, MA, USA), and pooled human serum from patients with ESKD (uremic serum) was obtained from more than 400 patients with ESKD undergoing hemodialysis at Shirasagi Hospital (Osaka, Japan). Blood samples were collected immediately before hemodialysis in a non-invasive manner. Normal and uremic serum were deproteinized by 3-times volume of methanol as much as serum and evaporated under a nitrogen stream at 50 °C; the resulting serum residues, normal serum residue (NSR) and uremic serum residue (USR), were used in the study^{3 (link)}. The present study was approved by the ethics committees of Shirasagi Hospital (IRB number J2012013) and Kyoto Pharmaceutical University (IRB number 08-04).

The human iPS-derived cells were treated with 1,25-Dihydroxyvitamin D3 (Sigma)10 (link) or rifampicin (Wako)25 (link), which are known to induce CYP 3A4 and P-gp, at a final concentration of 100 nM for 24 hr or 20 μM for 48 hr. Controls were treated with DMSO (final concentration 0.1%).