Ab186429

To positively identify exosomes, iPSC-MSC exosomes were denatured in SDS loading buffer for 5 min at 95 °C and then 10 μg of total protein was loaded and separated by SDS-PAGE on 10% gels. The proteins were transferred onto the PVDF membranes and stained with primary antibodies against CD63 (1:2000, catalog number Ab134045, Abcam, USA), Alix (1:5000, catalog number Ab186429, Abcam, USA), and TSG101 (1:1000, catalog number Ab125011, Abcam, USA) overnight at 4 °C after blocking with 4% skim milk. Then, the membranes were stained with goat anti-rabbit IgG HRP-conjugated antibody (1:2000, catalog number cw0103s, CWBIO) for 1 h and detected with Immobilon™ Western Chemiluminescent HRP substrate (Millipore, Billerica, MA, USA).

To evaluate the morphology, hADSC-Evs were suspended in 2.5% glutaraldehyde (1 mg/mL at protein concentration), dropped in carbon-coated copper grids, stained with 2% uranylacetate, dried, and examined by transmission electron microscopy (JEM 2100F TEM, Japan). To evaluate the size distribution, diluted hADSC-Evs (1 mg/mL at protein concentration) were subjected to Zetasizer Nano ZSP (Malvern Panalytical, Malvern, UK) and analyzed by the software equipped within the instrument. For western bloting, we detected extracellular vesicles biomarkers including CD63(1:1,000; Cat#ab134045, Abcam, Cambridge, UK), TSG101(1:1,000; Cat#ab125011, Abcam), Alix (1:1,000; Cat#ab186429, Abcam), and GAPDH (1:3,000; Cat#KC-5G5, Aksomics, Shang’hai, China).

A RIPA buffer containing a protease inhibitor (1×) was used to lyse the EV pellets (harvested as described above) at 95 °C for 5 min. Then, equal amounts of protein (20 μg) were resolved on a 10% sodium dodecyl sulfate-polyacrylamide gel, following which the protein bands were electrotransferred to a polyvinylidene difluoride membrane at 4 °C using a voltage of 100 V, a constant current of 0.1 A, and a run time of 1 h 20 min. Next, the membrane was incubated with 3% skim milk in Tween-containing Tris-buffered saline (TBS-T: 10 mM Tris, pH 8.0, 150 mM, and NaCl solution containing 0.05% Tween 20). Then, after three washes with TBS-T (10 min each), the membrane was incubated overnight at 4 °C with primary antibodies (diluted 1:1000 in 3% skim milk) targeting the following proteins: CD63 (ab217345, Abcam, Cambridge, UK), tumor susceptibility gene 101 protein (TSG101; ab30871, Abcam), and ALG-2-interacting protein X (Alix; ab186429, Abcam). Thereafter, the membrane was washed three times with TBS-T as described above and then incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG H&L (1:2000 dilution; ab205718, Abcam) for 1 h, at ambient temperature. Finally, after 5 min of signal development, the bands were visualized using an enhanced chemiluminescence detection system (Son et al., 2017 (link)).

Extracellular vesicles (EV) were isolated from a subset of HCM patients (n = 12) and healthy volunteers (n = 8) plasma samples and from iPSC-derived cardiomyocyte supernatants [37 (link)]. The purity of EV from both plasma and supernatant samples was tested by Western blotting in reducing conditions [36 (link)]. Total protein extracts (20 µg) were loaded on an 8–20% acrylamide gel, then transferred to a PVDF membrane and incubated in Intercept blocking buffer for 1 h at room temperature (LI-COR Biosciences, Lincoln, NE, USA) followed by primary antibodies against ALG-2 interacting protein X (Alix, part of Endosomal Sorting Complexes Required for Transport [38 (link)], ab186429, Abcam, Cambridge, UK), tumor susceptibility gene 101 (TSG101, an ESCRT component [39 (link)], ab30871 Abcam, Cambridge, UK) and tetraspanin CD81 [40 (link)] (MA5-17937, Thermo Fisher Inc., Waltham, MA, USA). After washing, the membranes were incubated for 1 h with secondary antibody (anti-mouse IgG IRDye-conjugated, LiCor). Images were acquired by Odyssey DLx and analyzed by Image Studio software (LI-COR Biosciences, Lincoln, NE, USA).

EVs from different groups were lysed with RIPA buffer. Protein quantification was carried out using Bicinchoninic acid assay (BCA) protein estimation kit (Thermofisher Scientific) according to the manufacturer's instruction. Proteins were extracted and loaded on 10% SDS‐PAGE gels, then transferred to PVDF membranes. After blocking with 3% non‐fat milk in Tris‐buffered saline with 0.1% Tween 20 (TBST) for 1 h at room temperature, the membranes were incubated overnight at 4°C with primary antibodies directed to: CD9 (Abcam ab92726, 1:1000 dilution), Alix (Abcam ab186429, 1:1000 dilution), TSG101 (Abcam ab125011, 1:000 dilution). After TBST washing, the membranes were incubated with corresponding secondary antibodies (CST 7076 S, 1:1000 dilution) for 1 h. Then, specific bands were detected by ECL reagent (Share‐Bio).

Immunoblotting was performed as previously described [6 (link)]. Proteins were detected using primary anti-CD9 (ab92726, Abcam, Cambridge, MA), anti-TSG101 (T5701, MilliporeSigma, St. Louis, MO), anti-Alix (ab186429, Abcam), anti-iNOS (13120S, Cell Signaling), anti-Arg-1 (93668S, Cell Signaling), anti-GAPDH (437,000, Thermo Fisher Scientific), anti-P (phospho)-Akt Ser473 (4060, Cell Signaling), anti-P-Akt Thr308 (13,038, Cell Signaling), and anti-Akt (4685, Cell Signaling) antibodies. Exposure of the resultant protein bands was performed with an ImageQuant LAS 4000 Luminescent Image Analyzer (GE Healthcare; Chicago, IL).