Dmem with high glucose

HEK293T cells (kindly provided by Hiroyuki Takatsu, Kyoto University) were plated on 6-cm dishes and cultured in DMEM with high glucose (Nacalai Tesque) supplemented with 10% fetal bovine serum (FBS). The cells were transfected with expression vectors for an EGFP fusion construct and a hemagglutinin (HA) fusion construct using Polyethylenimine Max and cultured for 24 h. The cells were then lysed in lysis buffer (50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid [HEPES]-KOH, pH 7.4, 100 mM KCl, 5 mM NaCl, 3 mM MgCl₂, 0.5% Triton X-100, 10% glycerol, and 1 mM dithiothreitol [DTT]) containing a protease inhibitor cocktail (Nacalai Tesque). The cell lysates were immunoprecipitated with GST–anti-GFP Nb prebound to glutathione–Sepharose 4B beads, and bound proteins were subjected to SDS–PAGE and immunoblotting analysis using anti-HA, anti-mRFP, anti-tRFP, or anti-GFP antibodies as described previously (Katoh et al., 2015 (link), 2016 (link); Nozaki et al., 2017 (link)).
The VIP assay was performed as described previously (Katoh et al., 2015 (link), 2016 (link)) with a slight modification; in this study, cells expressing EGFP-tagged and mChe-tagged proteins were lysed in the HMDEKN cell lysis buffer (10 mM HEPES, pH 7.4, 5 mM MgSO₄, 1 mM DTT, 0.5 mM EDTA, 25 mM KCl, 0.05% NP-40; Cole et al., 1998 (link)).

The VIP assay was performed as described previously (Katoh et al., 2015 (link), 2016 (link)). Briefly, HEK293T cells grown to ∼1.6 × 10⁶ cells on a six-well plate in DMEM with high glucose (Nacalai Tesque) supplemented with 5% fetal bovine serum (FBS) were transfected with expression vectors for EGFP and mChe/tRFP fusions (2 µg each) using Polyethylenimine Max (20 µg). After 24 h, the cells were lysed in 250 µl of lysis buffer (20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid–KOH, pH 7.4, 150 mM NaCl, 0.1% Triton X-100, and 10% glycerol) containing a protease inhibitor cocktail (EDTA-free; Nacalai Tesque). The lysates were mixed with 5 µl of GST–anti-GFP Nb beads at 4°C for 1 h. After washing three times with lysis buffer, the precipitated beads bearing fluorescent proteins were observed using an all-in-one-type fluorescence microscope (Biozero BZ-8000, Keyence) with a 20×/0.75 objective lens under constant conditions (sensitivity ISO 400, exposure 1/30 s for green; and sensitivity ISO 800, exposure 1/10 s for red) unless otherwise noted.

hACs were cultured as a micromass (1 × 10⁵ cells in 10 μL) on 12 well plates to evaluate ECM synthesis ability as described previously (Tateiwa et al., 2022 (link)). After a 3 h incubation, the chondrogenic basal media and each of the EV, BG sample or vehicle control (PBS) were added. Chondrogenic basal media consisted of DMEM with high glucose (Nacalai), 50 μg/mL L‐ascorbic acid, 40 μg/mL L‐proline (Wako), 1% ITS+ Premix (Corning, New York, NY, United States) and 1% AA, and each sample of sEVs, BG or vehicle control was added at a concentration of 5 × 10⁹ particles/mL for each of the EV group elements or at an equivalent volume for each of the BG or control groups. After 2 days of incubation, cell masses were collected by QIAzol (Qiagen, Hilden, Germany). RNA was isolated by Direct‐zol RNA Microprep kit (Zymo Research, Irvine, CA, United States) and cDNA was synthesized by using ReverTra Ace qPCR RT Master Mix with gDNA Remover (Toyobo) as described previously (Hanai et al., 2020 (link)). Gene expression (COL1A1 and COL2A1) was assessed by real‐time PCR on a Step One Plus system (Applied Biosystems, Carlsbad, CA, USA). Beta‐actin (ACTB) was used as an internal control gene for normalization and relative gene expression was calculated with the 2^−ΔΔ CT method. Primer sequences are listed in Table S1.