Pyruvate

HepG2 cells expressing ELuc and SLR3 were seeded in 96-well white clear-bottom plates (Nunc) at a density of 3 x 10⁴ cells/well. After an overnight incubation, the medium was replaced with DMEM without phenol red (Gibco-BRL) supplemented with 10% FBS, 4 mM glutamine, non-essential amino acids (Gibco-BRL), 1 mM pyruvate (FUJIFILM Wako Pure Chemical Corporation), 25 mM HEPES/NaOH (pH 7.0, Nacalai Tesque, Kyoto, Japan) and 300 μM d-luciferin potassium salt (Resem B.V., Lijnden, The Netherlands) with or without BU. Bioluminescence was recorded in real time for 5 s at approximately 30-min intervals in the absence or presence of the R62 long-pass filter (HOYA, Tokyo, Japan) at 37°C in 5% CO₂ atmosphere under saturated humidity using a microplate-type luminometer (WSL-1565 Kronos HT, ATTO, Tokyo, Japan). ELuc and SLR3 luminescence intensities were calculated as described previously (30 (link)).
BV2 cells bearing the reporter genes were harvested in PBS and homogenized by ultrasonic wave (UH-50 SMT-company, Tokyo, Japan) following the incubation of BU for 6 h. Luminescence obtained by addition of the luminescence substrate PGL100 reagent (TOYO INK, Tokyo) were measured in FlexStation3 (Molecular Device, CA).

A MAC6 vector was used to generate the 4CYPs-POR MAC. The structure of MAC6 contained a centromere from mouse chromosome 11, EGFP flanked by HS4 insulators, PGKneo, loxP-5’HPRT site, PGKpuro, and telomeres (S2 Fig) [27 (link)]. Hypoxanthine phosphoribosyl transferase (HPRT)-deficient Chinese hamster ovary (CHO; JCRB0218) hybrids containing only the MAC6 or the 4CYPs-POR MAC were maintained in Ham’s F-12 nutrient mixture (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum and 600 μg/mL G418. HepG2 cells (HB-8065, ATCC, USA) were maintained in Dulbecco’s modified Eagle’s medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum, non-essential amino acids (Wako), pyruvate (Wako), and penicillin-streptomycin (Wako).

ELuc-HepG2 or CYPs-ELuc-HepG2 cells were seeded in 96-well black clear bottom plates (Nunc, Wiesbaden, Germany) at a density of 1 × 10⁴ cells per well. After an overnight incubation, the medium was replaced with DMEM without phenol red (Gibco-BRL) but with 10% FBS (HyClone Laboratories), 4 mM glutamine, non-essential amino acids (Gibco-BRL), 1 mM pyruvate (FUJIFILM Wako Pure Chemical Corporation), 25 mM HEPES/NaOH (pH 7.0, Sigma–Aldrich), and 300 μM D-luciferin potassium salt (Resem B.V., Lijnden, The Netherlands) with or without compound. After incubation for 3 days in a CO₂ incubator, ELuc luminescence intensity in the cells was measured. The 96-well plate was set in a microplate-type luminometer (AB-2350 Phelios; ATTO, Tokyo, Japan) and luminescence was measured without disrupting the cells for 5 s. For the WST-1 assay, Premix WST-1 reagent (Takara Bio Inc., Shiga, Japan) was added to the remaining HepG2 cells and incubation was carried out for 1 to 2 h at 37 °C. Absorbance was measured at 450 nm (reference 620 nm) using the microplate reader Infinite 200 PRO (Tecan).