*2.2. Cell Culture*

Human embryonic kidney 293-derived 293A cells were maintained in Dulbecco's modified Eagle's medium (Nacalai Tesque, Kyoto, Japan) supplemented with 10% fetal bovine serum (Biowest, Nuaillé, France), 1% penicillin–streptomycin (Nacalai Tesque, Kyoto, Japan), 2 mM L-glutamine (Nacalai Tesque, Kyoto, Japan), and 1 × non-essential amino acid (Life Technologies, Carlsbad, CA, USA) at 37 ◦C in a humidified atmosphere of 5% (v/v) CO2 in air.

URAT1-expressing or mock plasmids were transfected into 293A cells using polyethylenimine "MAX" (PEI-MAX) (Polysciences, Warrington, PA, USA) as described previously [18], with some modifications. In brief, before transfection, 293A cells were seeded onto 12-well cell culture plates at a concentration of 0.92 × 10<sup>5</sup> cells/cm2. Then, 24 h after seeding, each plasmid vector was transiently transfected into the cells using PEI-MAX (1 μg of plasmid/5 μL of PEI-MAX/well). The medium was replaced with fresh medium 24 h after transfection.


**Table 1.** Key resources.

1 All antibodies were used at indicated dilutions in Tris-buffered saline containing 0.05% Tween 20 and 1% bovine serum albumin for 1 h at room temperature.

#### *2.3. Preparation of Protein Lysates and Immunoblotting*

Whole-cell lysates were prepared with cell lysis bu ffer A containing 50 mM Tris/HCl (pH 7.4), 1 mM dithiothreitol, 1% ( *w*/*v*) Triton X-100, and protease inhibitor cOmplete, EDTA free (Roche, Basel, Switzerland), and were treated with peptide *<sup>N</sup>*-glycosidase F (PNGase F) (New England Biolabs, Ipswich, MA, USA) as described previously [19]. Protein concentration was determined using the

PierceTM BCA Protein Assay Kit (Thermo Fisher Scientific, Kanagawa, Japan) with BSA as a standard, according to the manufacturer's protocol.

Whole-cell lysate samples were separated by SDS-PAGE and transferred to an Immobilon-P PVDF membrane (Millipore, Bedford, MA, USA) by electroblotting at 15 V for 60 min, as described previously [15]. Blots were probed with appropriate antibodies (Table 1), and the signals were visualized by chemiluminescence and detected using a multi-imaging Analyzer Fusion Solo 4TM system (Vilber Lourmat, Eberhardzell, Germany).

## *2.4. Confocal Microscopy*

For confocal laser scanning microscopic observation, 48 h after the transfection, 293A cells were fixed with 4% paraformaldehyde for 15 min at room temperature, and further processed according to previous studies [15,20]. In brief, the cells were treated with a fluorescent wheat germ agglutinin conjugate (WGA, Alexa Fluor® 594 conjugate; Thermo Fisher Scientific) to visualize plasma membranes, followed by nuclear staining using TO-PRO-3 Iodide (Molecular Probes, Eugene, OR, USA). Then, the cells were mounted in VECTASHIELD Mounting Medium (Vector Laboratories, Burlingame, CA, USA). To analyze the localization of EGFP-fused URAT1 protein, fluorescence was observed using the FV10i Confocal Laser Scanning Microscope (Olympus, Tokyo, Japan).

#### *2.5. Urate Uptake Assay Using URAT1-Expressing 293A Cells*

The urate uptake assay using URAT1-expressing 293A cells was conducted according to our previous studies [15,18] with minor modifications. In brief, 48 h after plasmid transfection, cells were washed twice with Cl−-free transport buffer (Buffer T2: 125 mM Na-gluconate, 4.8 mM K-gluconate, 1.2 mM KH2PO4, 1.2 mM MgSO4, 1.3 mM Ca-gluconate, 25 mM HEPES, 5.6 mM D-glucose, and pH 7.4) and pre-incubated in Buffer T2 for 15 min at 37 ◦C. The buffer was then exchanged with pre-warmed fresh Buffer T2 containing 5 μM [8-14C]-urate with or without test compound at the indicated concentrations (0, 0.1, 0.3, 1, 3, 10, 30, 100, or 300 μM), and the cells were further incubated for 20 sec; 1% DMSO was used as a vehicle control. The cells were subsequently washed five times with ice-cold Buffer T2 and then lysed with 500 μL of 0.2 M NaOH on ice with gentle shaking for 1 h. The lysates were neutralized with 100 μL of 1 M HCl. Then, the radioactivity in the lysate was measured using a liquid scintillator (Tri-Carb 3110TR; PerkinElmer, Waltham, MA, USA). The protein concentration was determined using the PierceTM BCA Protein Assay Kit. The urate transport activity was calculated as the incorporated clearance (μL/mg protein/min): (incorporated level of urate [disintegrations per minute (DPM)/mg protein/min]/urate level in the incubation mixture [DPM/μL]). URAT1-dependent urate transport activity was calculated by subtracting the urate transport activity of mock cells from that of URAT1-expressing cells.

Urate uptake was measured in the presence of several concentrations of each test compound to address their half maximal inhibitory concentration (IC50) values. URAT1-mediated transport activities were then expressed as a percentage of control (100%). Based on the calculated values, fitting curves were obtained according to the following formula using the least-squares method with Excel 2019 (Microsoft, Redmond, WA, USA):

$$\text{Predicted value} \left[ \% \right] = 100 - \left( \frac{\text{E}\_{\text{max}} \times \text{C}^{\text{n}}}{\text{EC}\_{\text{SO}}^{\text{n}} + \text{C}^{\text{n}}} \right) \tag{1}$$

where Emax is the maximum effect, EC50 is the half maximal effective concentration, C is the concentration of the test compound, and n is the sigmoid-fit factor. Finally, based on the results, the IC50 was calculated as described previously [15].

#### *2.6. Quantification and Statistical Analysis*

All statistical analyses were performed using Excel 2019 with Statcel4 add-in software (OMS publishing, Saitama, Japan). Different statistical tests were used for different experiments

as described in the figure legends, which include the number of biological replicates (*n*). Briefly, when analyzing multiple groups, the similarity of variance between groups was compared using Bartlett's test. When passing the test for homogeneity of variance, a parametric Tukey–Kramer multiple-comparison test for all pairwise comparisons was used. To investigate the inhibitory effect of each FA on URAT1 function (vs. vehicle control indicated as 100%) in the screening stage, one-sample *t*-test (one-sided) was conducted. Statistical significance was defined as *p* < 0.05 or 0.01.

Each experiment was designed to use samples required to obtain informative results and sufficient material for subsequent studies. No specific statistical test was used to pre-determine the sample sizes empirically determined in the current study. All experiments were monitored in a non-blinded fashion.
