Fig. 4

Myo-ApoEVs are generated during C2C12 myoblasts fusion process. a, b The fusion of C2C12 myoblasts increased over time. The immunofluorescence images of myosin staining of C2C12 myoblasts in fusion medium over time (a). The analysis of fusion index of C2C12 myoblasts in fusion medium over time (b). c, d The Apoptosis ratio of C2C12 myoblasts in the fusion medium was increased over time. The relative Annexin V expression of C2C12 myoblasts in fusion medium over time (c). The analysis of apoptosis ratio of C2C12 myoblasts in fusion medium over time (d). e, f Myo-ApoEVs are generated during C2C12 myoblasts fusion process over time. The representative immunofluorescence images of TO-PRO-3 staining of Myo-ApoEVs in fusion medium over time (e). The analysis of integrated fluorescence density of Myo-ApoEVs in fusion medium over time (f). g The size distribution of vesicles in supernatants of growth or fusion medium was examined by DLS. h The representative image of Myo-ApoEVs examined by SEM, wide-field and local magnification. i The expression of EVs-enriched protein TSG101 and Flotillin-1 with apoptosis-related proteins caspase-3 and cleaved caspase-3 of C2C12 myoblasts and Myo-ApoEVs examined by western blot. n = 3 per group; data were shown as mean ± SD; ns, not significant; the scale bar indicates 20 μm in the image a, e, and 500 nm in the image h; *P < 0.05, **P < 0.01, ***P < 0.001. DLS dynamic light scattering, SEM scanning electron microscope