Figure 3. Functional transfer of miR-146a via exosomes in vitro.

(a) Levels of miR-146a in the exosomal pellet derived from BMDCs that were treated with or without GW4869 and LPS (n=2). (b) miR-146a levels in Wt and Rab27 DKO BMDC-derived exosomal pellets (n=2). (c) Schematic of miR-146a exosome-transfer experiment where Wt or miR-146a−/− exosomes were isolated from BMDCs and transferred to recipient miR-146a−/− BMDCs. RNA was isolated after 24 h and the presence of miR-146a was assayed via qRT–PCR. (d) Relative levels of miR-146a in miR-146a−/− BMDCs given exosomes derived from Wt or miR-146a−/− BMDCs (n=4). (e) mRNA levels of miR-146a target, IRAK1, were measured from the same cells as in d via qRT–PCR (n=4). (f) Representative western blottings of IRAK1 and β-actin from miR-146a−/− cells given either Wt or miR-146a−/− exosomes. (g) IRAK1 protein levels were quantified using ImageJ software (n=2). (h) mRNA levels of miR-146a target, TRAF6, were measured in the same cells as in d via qRT–PCR. (i) Western blottings for TRAF6 and β-actin from miR-146a−/− BMDCs given either Wt or miR-146a−/− exosomes (n=2). (j) Western blotting results are quantified with ImageJ software. (k) Copy number of miR-146a in Wt and miR-146a−/− exosomes (n=3). Copy number is calculated based on a standard curve where a known amount of synthetic miR-146a was spiked into miR-146a−/− BMDC-derived exosome pellet followed by RNA isolation and qRT–PCR. (l) Copy number of miR-146a was measured via qRT–PCR in miR-146a−/− recipient BMDCs that received either Wt or miR-146a−/− exosomes (146a−/− BMDC+Wt exos and 146a−/− BMDC+146a−/− exos), as well as in Wt and miR-146a−/− donor BMDCs (n=3). Average copy number is displayed above. Copy number is calculated based on a standard curve where a known amount of synthetic miR-146a was spiked into miR-146a−/− BMDC pellet followed by RNA isolation and qRT–PCR. Data represent two independent experiments and are presented as the mean±s.d. (error bars). *P<0.05, Student's t-test.