Tsg101

Total proteins were extracted from atrial samples using radioimmunoprecipitation assay buffer plus phosphoprotease inhibitors (ASPEN Biotechnology, China). The same amount (40 μg) of extracted protein was separated by electrophoresis in SDS-PAGE gels and transferred to polyvinylidene difluoride membranes. Membranes were blocked with 5% blocking buffer for 1 h at room temperature and incubated overnight at 4°C with primary antibodies against CD63 (Biorbyt, England), CD81 (Abcam, USA), TSG101 (Sigma-Aldrich, Germany), Rab27a (Sanying Biotechnology, China), collagen I (Novusbio, USA), collagen III (Abcam, USA), matrix metalloproteinase (MMP)-2 (Bioss, USA), MMP-9 (Bioss, USA), tissue inhibitor of metalloproteinase 3(TIMP3) (Lsbio, USA), and transforming growth factor-β1 (TGF-β1) (Sanying Biotechnology, China). The membranes were washed three times with tris-buffered saline with 0.1% Tween® 20 and then incubated with horseradish peroxidase-conjugated anti-rabbit secondary antibody (ASPEN Biotechnology, China) for 1 h at room temperature. The blots were exposed with an ECL Detection Kit (ASPEN Biotechnology, China). The expression levels of the proteins were determined and normalized to the relative intensity of GAPDH using image analyzer software (AlphaEase FC, USA).

Protein content of each SEC fraction was measured by micro BCA protein assay (Thermo Fisher Scientific), and input from the separate SEC fractions was adjusted accordingly to obtain equal loading of every SEC fraction onto the 4–12% SDS PAGE gel (Bio Rad). Protein concentration was eventually determined by a precipitation assay with trichloroacetic acid (Sigma, Kanagawa, Japan). After transfer to a nitrocellulose membrane (Bio Rad), the membrane was cut into two parts to allow for staining for different targets simultaneously. The membrane was blocked with PBS containing 5% (w/v) bovine serum albumin and then incubated with CD9 (Santa Cruz Biotechnology, Dallas, TX, USA, sc52519, 1:1000) and CD63 (BD Biosciences, San Jose, CA, USA, 556019, 1:1000). Antibody binding was visualized with anti-mouse IgG coupled to horse radish peroxidase at a 1:5000 dilution. Subsequently, the membranes were stripped by incubating with 1% NaN3 for an hour, and after blocking, incubated with CD81 (Santa Cruz Biotechnology, Santa Cruz, Dallas, TX, USA, SC9158, 1:1000) and TSG101 (Sigma, St. Louis, MI, USA, T5701, 1:1000).

Following antibodies were used: anti-GFP rabbit, actin, and Tsg101 (Sigma-Aldrich); Alix and CHMP3 (Santa Cruz Biotechnology); and monoclonal and polyclonal anti-B5 (VMC-20 and R182, respectively) were kind gift of GH Cohen (University of Pennsylvania, Philadelphia, PA, USA). CHMP1A, CHMP2B, CD63, GM130, and TGN46 (Abcam); CHMP1B (ProteinTech Group); EEA1 and Lamp1 (Cell Signaling). Anti-L1 mouse monoclonal antibody (clone 7D11) was purified from a hybridoma cell line that was provided by B. Moss (National Institutes of Health) with permission of A. Schmaljohn (University of Maryland). IRDye-coupled secondary antibodies and Alexa-secondary antibodies were purchased from Invitrogen/Thermo Fisher Scientific.

MSCs were isolated from bone-marrow aspirates of four healthy individuals after informed consent as described before [6 (link)]. Approval of the Ethical Committee of the University Hospital Essen was obtained (12-5295-BO, 17.01.2013 and 25.06.2018). Cell culture supernatants (conditioned media; CM) were collected for EV purification by differential centrifugation and PEG precipitation as recently described [15 (link)]. For details, see supplementary information. MSC-EV fractions were studied (i) by nanoparticle tracking analysis on the ZetaView (Particle Metrix, Meerbusch, Germany) to define size and particle concentration (Table S1) [16 (link)], (ii) by protein assay (Thermo Scientific, Darmstadt, Germany) to define the protein concentration (Table S1) and (iii) by SDS PAGE and western blot to verify the expression of components associated with EVs including TSG101 (Sigma-Aldrich, St. Lois, MO, USA), Syntenin (clone EPR8102; Abcam, Cambridge, UK), CD81 (clone 5A6; Biolegend, San Diego, CA, USA), CD9 (VJ1, kindly provided by Francisco Sánchez-Madrid) and the absence of cytochrome C (clone 6H2.B4; Biolegend) to exclude cellular protein contamination as previously recommended [17 (link),18 (link)] as minimal requirement for definition of EVs (Figure S1).