Bovine serum albumin (bsa)

Worms were lysed in 20 mM Tris/HCl buffer (pH 7.4) containing 1.0 % Triton X-100, 150 mM NaCl, and cOmplete™ Protease Inhibitor Cocktail (F. Hoffmann-La Roche AG, Basel, Switzerland). After centrifugation at 12,000 rpm for 10 min, the supernatants were obtained. Protein concentration was measured using BCA Protein Assay Kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Supernatants containing 20 µg of proteins were separated by a 15 % SDS-PAGE gel under reducing conditions and blotted onto a PVDF membrane. The membrane was blocked with 5.0 % bovine serum albumin (Nacalai Tesque) for the detection of phosphorylated proteins and 1.0 % skim milk (Wako Pure Chemical Industries) for other proteins. The primary antibody was mouse anti-GFP IgG (1/1,000; Sigma-Aldrich Corporation, St. Louis, MO, USA). The secondary antibody was horseradish peroxidase-conjugated anti-mouse IgG (1/4,000; MEDICAL & BIOLOGICAL LABORATORIES CO., LTD., Nagoya, Japan). Detection of the target proteins was carried out using West Pico Chemiluminescent Kit (Thermo Fisher Scientific) and LAS Image Analyzer (Fuji Film Corporation, Tokyo, Japan).

To assess the uptake of plasma sEV into RASM, they were treated with WsEV or SsEV, which
were labeled with PKH67 green fluorescence dye (Sigma Aldrich). WsEV or SsEV were reacted
with PKH67 (4 µM, RT, 3 min), which was stopped with the same volume of
sterilized PBS containing 10% bovine serum albumin (Nacalai Tesque). To wash the excess
dye, the sEV-PKH67 solution was taken into a tube containing a sterilized 0.971 M sucrose
solution in the bottom and ultracentrifuged (164,071 × g, 4°C, 35 min). The supernatants
were removed by pipetting and the pellets were resuspended in sterilized PBS. RASM were
treated for 2 hr with PKH67-labeled sEV (1.0 × 10⁸particles/ml) or PKH67-reacted PBS (Cont) and fixed with 4%
paraformaldehyde (FUJIFILM Wako Pure Chemical) (4°C, 10 min). Nuclei were stained with 4’,
6-diamidino-2-phenylindole (DAPI, Dojindo Laboratories, Kumamoto, Japan) (RT, 10 min) and
photographed by using a fluorescence microscope (BX-51) with a digital camera (DP74) and
CellSens standard dimension ver. 1.18 software (Olympus). Fluorescence density in a single
cell was measured using an ImageJ software (National Institutes of Health, Bethesda, MD,
USA).

EVs were lysed in lysis buffer containing 50 mM Tris-HCl (pH 7.5), 50 mM sodium fluoride, 0.15 M NaCl, 1% Triton X-100, 1 mM EDTA, 30 mM sodium pyrophosphate, 1 mM phenyl-methanesulfonyl fluoride, and 2 mg/ml aprotinin. Protein concentrations were determined by bicinchoninic acid (BCA) assay. Laemmli sample buffer containing 0.1 M Tris-HCl buffer (pH 6.8), 1% SDS, 0.05% mercaptoethanol, 10% glycerol, and 0.001% bromophenol blue was added and boiled (95˚C, 5 min). Proteins (5 µg/lane) were separated by reducing 8% (weight/volume) polyacrylamide gel electrophoresis and electroblotted on nitrocellulose blotting membranes (Cytiva). The membranes were blocked for 1 h at room temperature in 0.1% Tween-20/Tris-buffered saline containing 1% bovine serum albumin (all from Nacalai Tesque, Inc.) and incubated overnight at 4˚C in the presence of anti-CD9 antibodies (1:100,000 dilution; cat. no. sc-9148; Santa Cruz Biotechnology, Inc.). After the membranes were washed and then incubated (25˚C, 1 h) with the appropriate HRP-conjugated goat anti-rabbit secondary antibody (1:10,000 dilution; cat. no. 12-348; EMD Millipore), the bands were visualized using TMA-6 chemiluminescence reagent (Lumigen, Inc.; Beckman Coulter, Inc.). The band images were obtained using FUSION Solo S (Vilber-Lourmat).

CHO-K1, CHO/EpCAM and Caco-2 cells were isolated using 0.25% trypsin and 1 mM ethylenediamine tetraacetic acid (EDTA; Nacalai Tesque, Inc.) treatment. The cells were treated with EpMab-37-mG_2a-f, or blocking buffer [0.1% bovine serum albumin (BSA; Nacalai Tesque, Inc.) in phosphate-buffered saline (PBS)] (control) for 30 min at 4°C. Subsequently, the cells were incubated in Alexa Fluor 488-conjugated anti-mouse IgG (1:2,000; cat. no. 4408; Cell Signaling Technology, Inc.) for 30 min at 4°C. Fluorescence data were collected using the SA3800 Cell Analyzer and analyzed using SA3800 software ver. 2.05 (Sony Corporation).