Release of EVs after LIPUS treatment in vitro and in vivo. (A) EVs were isolated from the myocardial tissue of iDCM + LIPUS and iDCM mice on day 42. (B) Representative TEM of isolated EVs from the myocardial tissue (scale bars = 100 nm). (C, D) Representative blots of EVs marker protein (Alix, CD63) and contaminant protein (Calnexin, ApoA1) in EVs purified. CD4⁺ T-cell lysates were used as a control for Alix, CD63 and Calnexin (Alix: P = 0.0229；CD63: P = 0.0427, N = 5 independent repeats). (E) Representative results of NTA demonstrating size distributions and numbers of EVs (P = 0.0012, n = 5). (F, G) The relative protein expression of cellular markers in EVs were measured by Western Blot (Cav-3: P = 0.0214; CD105: P < 0.0001, N = 5 independent repeats). (H) Cellular origin analysis of EVs isolated from myocardial tissue of iDCM + LIPUS and iDCM mice (CD144⁺: P = 0.0012; CD105⁺: P = 0.0047, n = 5). (I) Morphology of HCAECs-derived MVBs observed by TEM (scale bars = 500 nm). Quantification the number of MVBs (P < 0.0001, n = 5). (J) Measured total EVs in conditioned medium per cell by NTA (P < 0.0001, n = 5). Measured total EV proteins in the conditioned medium by bicinchoninic acid assay (BCA) (P = 0.0009, n = 5). Results are expressed as mean ± SD. Comparisons of parameters were performed with student's t-test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.