To establish if AGO-2-miRNA complexes were present in the PF samples, AGO-2 immunoprecipitation (IP) was performed (Figure 4A) and followed by miRNA RT-qPCR analyses. The majority of our miRNAs were found conjugated to AGO-2 (Figure 4B). The relative expression of the individual miRNAs conjugated to AGO-2 was variable (Figure 4B), and it did not follow the same trends of exosomal miRNA expression (Figure 3A). Interestingly, the majority of studied miRNAs were co-expressed in exosomes and AGO-2 complexes, suggesting the possibility that PF exosomes contain AGO-miRNA complexes. In line with this, AGO-2 was detected (by western blot analysis) in the PF exosome preparations (Figure 4C). Moreover, AGO-2-IP of PF exosomes, followed by PCR for a randomly selected subset of the miRNAs expressed in the AGO2 complexes, confirmed the presence of AGO-2-miRNA complexes in the exosomal compartment of human PF (Figure 4D). Also in this case, the relative expression of the individual exosomal miRNAs conjugated to AGO-2 was variable, with let-7b-5b appearing particularly enriched in the AGO-2 complex (Figure 4D). Dicer was also present within the PF exosomes (Figure 4E). Similarly to PF, plasma exosomes also contained both Dicer and AGO-2 (Supporting Materials and Methods). When the exosome membrane was not previously destroyed by sonication, the exosomal miRNAs (let-7b-5p and miR-122) and proteins (DICER and AGO2) were resistant to proteinase K (PK) and RNase A digestion (Figure S3). This favors the hypothesis that the miRNAs, DICER and AGO-2 were indeed encapsulated inside the exosomes, rather than being merely co-precipitated with them. Taken together, the above data provide evidence that exosomes contain miRNAs that are co-expressed and possibly physically associated with the RISC. It is therefore possible that exosomes deliver miRNAs together with a RISC machinery ready to act in recipient cells, thus immediately eliciting expressional changes commanding functional responses.Figure 4PF Exosomes Contain DICER and AGO-2 Protein(A) Validation by immunoblotting of AGO-2 immunoprecipitation (IP) performed on human PF samples using AGO-2 antibody. Mouse non-specific IgG antibody was used as control for the IP. ECs were used as positive control (CTRL). (B) The miRNA expression after AGO-2 IP is presented as fold enrichment relative to IgG; mean + SEM; n = 5. (C) AGO-2 in exosomes enriched from PF samples (representative western blot images). (D) AGO-2 IP was performed on exosomes enriched from PF samples. miRNA expression is expressed as fold enrichment in the AGO-2 IP relative to IgG; mean + SEM; n = 2. (E) Representative western blot images of DICER protein incorporated in the exosomes.