Genistein

Daidzein, daidzin, daidzin 6″-O-acetate, daidzin 6″-O-malonate, genistein, genistin, genistin 6″-O-acetate, genistin 6″-O-malonate, glycitein, glycitin, glycitin 6″-O-acetate, and glycitin 6″-O-malonate were purchased from FUJIFILM Wako Pure Chemical Corp (Osaka, Japan). CMS; dimethyl sulfoxide (DMSO); Gelrite™; indole-3-butyric acid (IBA); ascorbic acid; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; methyl jasmonate (MJ); and phosphate-buffered saline were purchased from Sigma-Aldrich Co., LLC (St. Louis, MO, USA). Mass-grade formic acid (FA), acetonitrile, methanol, and water were purchased from Thermo Fisher Scientific (Waltham, MA, USA). The other chemicals employed in this study were of American Chemical Society grade or higher. The Sinhwakong soybean variety (an elite cultivar) was supplied by the National Institute of Crop Science (Cheonju, Republic of Korea).

For treatment with antibodies, HeLa or K562 cells were incubated with anti-CD33 (15 µg/mL; Southern Biotech, Birmingham, AL, USA), anti-Tim-1 (20 µg/mL; BioLegend, San Diego, CA, USA), or anti-Tim-4 antibody (20 µg/mL; Novus Biologicals, Centennial, CO, USA) for 1 h at 37 °C. The prepared EV samples were then inoculated onto the cells in the presence of the antibodies. After incubation for 4 h at 37 °C, the inoculum was removed and the cells were maintained in MEM-1%FBS for 72 h before the luciferase assay.
For treatment with various entry inhibitors, HeLa cells prepared in the 96-well plate were incubated with Pitstop 2 (30 µM; Merck), LY294002 (5 µM; FUJIFILM Wako Pure Chemical Corporation), dynasore (80 µM; Tokyo Chemical Industry Co., Tokyo, Japan), genistein (150 µM; FUJIFILM Wako Pure Chemical Corporation), or 5-[N-ethyl-N-isopropyl] amiloride (EIPA: 100 µM; Cayman Chemical) for 30 min at 37 °C. The prepared EV samples were then inoculated onto the cells in the presence of entry inhibitors. Four hours later, the cells were rinsed with 0.5 M NaCl–0.2 M acetic acid solution and then maintained in MEM–1%FBS for 72 h before the luciferase assay.

ED cells were kindly provided by Dr M. Maeda (Kyoto University, Japan), and S1T cells were kindly provided by Dr N. Arima (Kagoshima University, Japan). MT-2 and Jurkat cells were obtained from the Fujisaki Cell Center, Hayashibara Biochemical Laboratories (Okayama, Japan) [26 (link)]. All cells were maintained in RPMI 1640 medium (Sigma-Aldrich Co., St. Louis, MO, USA) supplemented with 10% fetal bovine serum (Sigma-Aldrich Co., lot no. 13C491) containing 100 U/mL penicillin G and 100 μg/mL streptomycin (Life Technologies, Carlsbad, CA, USA). Each cell line was seeded (1 × 10⁵ cells/mL, 90 μL/well) into a 96-well plate containing RPMI 1640 medium. After incubation at 37 °C for 24 h in an atmosphere containing 5% CO₂, samples were added (10 μL/well) to the cells and incubated for an additional 72 h. Subsequently, the inhibition of cell proliferation was determined using a 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt (WST-8) assay kit (Dojindo, Kumamoto, Japan). Viable cells convert the tetrazolium salt in WST-8 to highly water-soluble formazan, which is monitored by measuring the absorbance at 450 nm using a microplate reader (Multiskan FC, Thermo Fisher Scientific, Waltham, MA, USA). Genistein (Wako, Osaka, Japan) was used as a positive control [21 (link)].