Fig. 4

RNA-binding proteins (RBPs) mediate miR-146a/155 packaging into macrophage-derived EVs. (A) Direct interactions between the miR-146a/155 sequences and RBP motifs were predicted using RBPDB analysis (threshold 0.5). (B) Western blot results showing RBP (PTBP1 and NONO) expression in macrophage-derived EVs. C and D. Western blot and qPCR results showing PTBP1 and NONO expression levels in macrophages 48 h after transfection with specific siRNAs. E. The levels of FITC-labeled PTBP1 and cy5-labeled miR-146a exhibit alterations throughout the polarization process of M1 macrophages. PTBP1, an RNA-binding protein for miR-146a, exhibited a shift in its expression pattern from nuclear localization to clustering on the cell membrane upon macrophage polarization. The fluorescence signal emitted by cy5 exhibits a gradual decline as the macrophage undergoes polarization within the cellular environment. F. Levels of FITC-labeled NONO and cy5-labeled miR-155 exhibit alterations throughout the polarization process of M1 macrophages. NONO, an RNA-binding proteins for miR-155, and miR-155 exhibit a gradual decline as the macrophage undergoes M1 polarization within the cellular environment. G. Co-localization analysis of PTBP1 and miR-146a reveals a gradual strengthening of the co-localization signal as macrophages polarize towards the M1 phenotype. H. Co-localization analysis of NONO and miR-155 reveals a gradual strengthening of the co-localization signal as macrophages polarize towards the M1 phenotype. I. Western blot analysis of PTBP1 and NONO expression in samples derived by biotinylated miR-146a/155 pulldowns performed with whole cell lysis, or EV lysis of macrophages, and the indicated biotinylated miR-146a/155 or mutated miR-146a/155. biotinylated poly (G) was used as a negative control. J. AECs were co-cultured with macrophages concurrently transfected with Cy5-miR-146a/155 and specific siRNAs targeting PTBP1 or NONO for 48 h. Fluorescence microscopy was used to detect red fluorescence signals in AECs (scale bar = 100 μm). ns, not significant, *P < 0.05, **P < 0.01