Fig. 4. The results of the CD9-CIBER screening and comparison with those of CD63-CIBER screening.

a The pair-wise plot of z-REs obtained from CD63/CD9-CIBER screen for 10,410 genes. The shaded region contains the genes showing the z-RE difference between CD63-CIBER and CD9-CIBER within the mean ±2 SD. b Gene-GO term association matrix where black dots are plotted at the intersection if genes are annotated to the terms. The heatmap at the top shows z-RE of each gene annotated to GO terms grouped as OxPhos in Fig. 3i, whose -log₁₀(p-value) (two-tailed Fisher’s exact test with Holm correction) are displayed as a heatmap at the left. Only genes with z-REs less than −1.65 in either screening are displayed. c Volcano plot of enrichment score of each gene set calculated via GSEAPreranked after ranking all the screened 10,410 genes. The dashed line shows adjusted p-value of 0.25 calculated by two-tailed Kolmogorov–Smirnov test with Benjamini–Hochberg correction. In this data representation, KO or inhibition of components annotated to gene sets in the upper left/upper right area is predicted to downregulate/upregulate the release of each subpopulation of sEVs, respectively. d, e Relative abundance of CD63 and CD9 on the surface of sEVs after treatment with rotenone (an inhibitor of mitochondrial complex I) at 10 nM (d) or concanamycin A (ConA, a V-type ATPase inhibitor) at 1 nM (e) for 24 h. sEVs were captured on Tim4-immobilized wells and detected using either anti-CD63 antibody or anti-CD9 antibody conjugated with horseradish peroxidase. Vehicle: 0.1% DMSO. Error bars represent ±SEM of biological replicates (n = 3). f Proposed mechanisms of selective change of the release of CD63⁺ sEVs associated with lysosomal perturbation. Treatment of cells with ConA neutralizes lysosomes, and the contents of MVB tend to evade lysosomal degradation, leading to increased release of “exosomal” CD63⁺ sEVs. Conversely, treatment of cells with rotenone enhances the activity of lysosome, leading to decreased release of exosomal CD63⁺ sEVs. On the other hand, CD9⁺ sEVs are rather “ectosomal”, so their release is less affected. p: two-tailed Welch’s t-test with Holm correction. *p < 0.05, **p < 0.005, ***p < 0.0005. NS, not significant.