*2.5. Immunoblotting*

The expression of ABCC11 protein in plasma membrane vesicles was examined by immunoblotting, as described previously [4,9] with minor modifications. Briefly, the prepared samples were electrophoretically separated on poly-acrylamide gels and transferred to a Hybond ® ECLTM nitrocellulose membrane

(GE Healthcare, Buckinghamshire, UK) by electroblotting at 15 V for 70 min. After blocking by Tris-buffered saline containing 0.05% Tween 20 and 5% skim milk (TBST-skim milk) at 4 ◦C overnight, blots on the membrane were probed with a rat monoclonal anti-ABCC11 antibody (M8I-74; Abcam, Cambridge, MA, USA; diluted 200 fold) and a rabbit polyclonal anti-Na+/K<sup>+</sup>-ATPase α antibody (sc-28800; Santa Cruz Biotechnology, Santa Cruz, CA, USA; diluted 1000 fold), followed by incubation with a goa<sup>t</sup> anti-rat immunoglobulin G (IgG)–horseradish peroxidase (HRP) conjugated antibody (NA935V; GE Healthcare; diluted 2000 fold) and a donkey anti-rabbit IgG–HRP conjugated antibody (NA934V; GE Healthcare; diluted 3000 fold), respectively. All antibodies were used in TBST-skim milk. HRP-dependent luminescence was developed using the ECLTM Prime Western Blotting Detection Reagent (GE Healthcare) and detected using a multi-imaging Analyzer Fusion Solo 4TM system (Vilber Lourmat, Eberhardzell, Germany).

#### *2.6. Vesicle Transport Assay*

The inhibitory effects of the various target extracts and compounds on the ABCC11 function were examined using the in vitro vesicle transport assay, a well-established method to quantitatively evaluate ABC transporter function [13]. For this purpose, the ATP-dependent transport of [1,2,6,7-3H(N)]- dehydroepiandrosterone sulfate (DHEA-S) (PerkinElmer, Waltham, MA, USA), which is an ABCC11 substrate [7], into the ABCC11-expressing and control plasma membrane vesicles was quantified following our previous study [9] with some minor modifications in the rapid filtration technique, as described below.

In brief, the plasma membrane vesicles (0.25 mg/mL or indicated concentrations) were incubated with [1,2,6,7-3H(N)]-DHEA-S (100 nM or indicated concentrations) in a reaction mixture (total 20 μL: 10 mM Tris/HCl, 250 mM sucrose, 10 mM MgCl2, 10 mM creatine phosphate, 1 mg/mL creatine phosphokinase, 50 mM ATP or AMP as a substitute of ATP, and pH 7.4) for 5 min at 37 ◦C, either without (i.e., with only vehicle control) or with the individual target fractions/authentic chemicals at the indicated concentrations. As the vehicle control, 1% water was used for plant extracts; 1% methanol (Nacalai Tesque) or 1% dimethyl sulfoxide (DMSO; Nacalai Tesque) was used for the individual target fractions, as described below. Since stock solutions of authentic chemicals were prepared with DMSO at 10 mM, 1% DMSO was employed as the vehicle control for them. After incubation, the reaction mixture was mixed with 980 μL of an ice-cold stop bu ffer (2 mM EDTA, 0.25 M sucrose, 0.1 M NaCl, 10 mM Tris-HCl, and pH 7.4) and rapidly filtered on a membrane filter (MF-Millipore Membrane (HAWP02500; Millipore, Tokyo, Japan) for extract screening or Whatman ™ Grade GF/F Glass Microfiber Filter Paper (GE Healthcare) for the other experiments). After washing with 5 mL of the ice-cold stop bu ffer three times, the plasma membrane vesicles trapped on the membrane filter were dissolved in Clear-sol II (Nacalai Tesque). Then, the radioactivity incorporated into the plasma membrane vesicles was measured with a liquid scintillator (Tri-Carb 3110TR; PerkinElmer).

The transport activity in each group was calculated as the incorporated clearance (μL/mg protein/min = incorporated level of DHEA-S (disintegrations per minute (DPM)/mg protein/min)/ DHEA-S level in the incubation mixture (DPM/μL)). ATP-dependent DHEA-S transport was calculated by the di fference in transport activity with and without ATP. Similarly, ABCC11-mediated DHEA-S transport activity was calculated by subtracting the ATP-dependent DHEA-S transport activity of control plasma membrane vesicles from that of ABCC11-expressing ones. Unless otherwise indicated, effects of the target fractions/compounds on the ATP-dependent DHEA-S transport activity were also examined for the control plasma membrane vesicles.

#### *2.7. Fractionation of Soybean (Glycine max) Extract*

Medium-pressure liquid chromatography (MPLC) was conducted using a dual channel automated flash chromatography system (EPCLC-W-Prep 2XY; YAMAZEN, Osaka, Japan), as described below. All the eluates were evaporated to dryness and then stored at −20 ◦C. They were reconstituted in an appropriate solvent before use in the vesicle transport assay for the evaluation of ABCC11-inhibitory activities and/or chemical characterization by mass spectrometry (MS) analysis.

The water extract of dry soybeans was separated into 12 fractions (Fr.#1-12) by MPLC on an octadecyl-silica (ODS) column (DispoPackAT ODS-25; particle size 25 μm, column size 120 g, i.d. 40 × 188 mm; YMC, Kyoto, Japan). The separation was performed in the linear gradient elution mode with solvent A (0.2% formic acid in water) and solvent B (0.2% formic acid in acetonitrile) (solvent A:solvent B (*v*/*v*): 0–5 min 95:5; 5–25 min 95:5 to 0:100; and 25–35 min 0:100) at a flow rate of 40 mL/min, with UV monitoring at 265 nm using an equipped UV detector. Each fraction was reconstituted (10 mg/mL) in an appropriate solvent (i.e., water for Fr.#1 and Fr.#2, 50% methanol for Fr.#3-11, and methanol for Fr.#12) before use.

Among the 12 fractions, Fr.#11 (the target fraction reconstituted in 50% methanol) was further subjected to MPLC over an ODS column (RediSep ODS GOLD; 5.5 g media, 20–40 μm spherical; Teledyne Isco, Lincoln, NE, USA) in the stepwise elution mode using a mixture of the same A and B solvents (solvent A:solvent B (*v*/*v*): 0–2 min 80:20; 2–9 min 50:50; and 9–17 min 0:100) at a flow rate of 15 mL/min with UV monitoring at 254 nm. This gives three subfractions (Fr.#11-1 to Fr.#11-3) plus a dominant peak eluted from 3.0 to 5.2 min. The dominant peak was collected and then further separated in the same column with a linear gradient of 10–50% of solvent B in solvent A to give three more subfractions (Fr.#11-4 to Fr.#11-6).

Finally, to further separate ABCC11-inhibitory ingredients, Fr.#11-5—the most active subfraction among Fr.#11-1 to Fr.#11-6 in terms of ABCC11 inhibition—was purified by a recycling preparative HPLC system (LaboACE LC-5060; Japan Analytical Industry, Tokyo, Japan) equipped with a gel permeation column (JAIGEL-GS310; i.d. 20 × 500 mm; Japan Analytical Industry), using methanol as a mobile phase at 5 mL/min and with refractive index monitoring and UV monitoring at 254 nm. In brief, Fr.#11-5 was separated by the recycling mode for 120 min. Then, Fr.#11-5-1 and Fr.#11-5-2 were collected from 123 to 126 min and from 160 to 176 min, respectively. All the wastes were collected and further processed as Fr.#11-5-3. Additionally, all the subfractions were evaporated to dryness and then stored at −20 ◦C. They were reconstituted in DMSO (2 mg/mL) before use.

## *2.8. Chemical Characterizations*

For the qualitative determination of the isolated compounds, chromatographic separations, and subsequent MS (or MS/MS) analyses were carried out with an LC-quadrupole time-of-flight (Q-TOF)-MS/MS system consisting of an HPLC instrument (Agilent 1100 Series equipped with a diode array and multiple wavelength detector (DAD) (G1316A); Agilent Technologies, Santa Clara, CA, USA) coupled with an Agilent 6510 Q-TOF (Agilent Technologies). The chromatographic conditions and MS setting were drawn from our previous study [14] with some minor modifications. Briefly, the separation was performed on a Zorbax Eclipse Plus C18 column (2.1 × 100 mm; Agilent Technologies) maintained at 40 ◦C under gradient mobile conditions with a mixture of solvent C (0.1% formic acid in water) and solvent D (acetonitrile) (solvent C:solvent D (*v*/*v*): 0–8 min 95:5 to 5:95, and 8–12 min 5:95) with a flow rate of 0.5 mL/min. The detection range of the DAD was set from 190 to 400 nm, and the MS detection system operated in the positive ionization mode at an MS scan range of *m*/*z* 100–1700. Peak analysis was performed using the Agilent MassHunter Workstation software (version B.03.01; Agilent Technologies).

#### *2.9. Calculation of the Half-Maximal Inhibitory Concentration Values*

To calculate the IC50 value of genistein against DHEA-S transport by ABCC11, the DHEA-S transport activities were measured in the presence of genistein at several concentrations. The ABCC11- mediated DHEA-S transport activities were expressed as a percentage of the control (100%). Based on the calculated values, fitting was carried out with the following formula using the least-squares methods in Excel 2019 (Microsoft, Redmond, WA, USA), as described previously [15]:

$$\text{Predicted value } [\![\!0]\!] = 100 - \left( {}^{\text{E}\_{\text{max}}} \times {}^{\text{C}}\!/ {}^{\text{E}}\!/ {}^{\text{C}}\!/ {}^{\text{a}}\!/ {}^{\text{a}}\!/ {}^{\text{a}}\right) \tag{1}$$

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. IC50 was calculated based on these results.
