Otection provided by Ago2 complexes to the various miRNAs in the MVs was different. This finding may raise an important question about the fate of the circulating miRNAs in the cell-derived MVs. Because Ago2 is also a key effector of miRNA function, our results may imply that only the secreted miRNAs that are associated with Ago2 complexes in the cell-derived MVs are stable and have biological function after they enter into the recipient cells, whereas the non-Ago2 complex-bound miRNAs in the MVs may be simply Indolactam V degraded in the recipient cells. Our results further showed that thestability of a circulating miRNA in the cell-derived MVs is positively correlated with the degree of its association with Ago2 complexes. As shown in the in vitro digestion assay with RNase A (Figure 3), Ago2 complex-associated miR-16 was significantly resistant to RNase A compared with free miR-16, which was rapidly degraded by RNase A. These results suggest that certain cell secreted miRNAs are pre-loaded with Ago2 complexes in MVs released by origin cells and can be delivered into recipient cells where they start inhibiting their targets. In other words, the secreted miRNAs in MVs are already functionally equipped with Ago2 and can directly execute their roles in the recipient cells. Therefore, MV-delivery of secreted miRNAs provides a new mechanism for cell-to-cell communication. The Ago2/miRNA complexes are also highly protease-resistant, as miRNA remained stable in the cell lysates for over a week (data not shown). TheAgo2 Complexes Protect Secreted miRNAsFigure 5. Enhancement of both the association of miRNAs with Ago2 complexes in cell-derived MVs and the resistance of miRNAs to RNaseA under various physiological conditions. A) The apoptosis of HeLa cells induced by serum starvation and TNFa treatment for 24 h, respectively. B) Relative BIBS39 supplier levels of total and Ago2 complex-associated miR-16, miR-30a, miR-223 and miR-320b in the MVs derived from HeLa cells with or without apoptotic reagent treatment. Note that, although the total miR-16 levels are not changed, the percentage of miR-16 associated with Ago2 complexes is increased under apoptosis induced by either serum starvation or TNFa treatment. C) The resistance of miR-16 in 
HeLa cell-derived MVs to RNaseA. D) The differentiation of HL60 cells induced by 20 mM ATRA 
for 48 h. E) Relative levels of total miR-16, miR-30a, miR-223, miR-320b and the levels of these miRNAs associated with Ago2 complexes in the MVs derived from HL60 cells with or without induced by ATRA. Note that, although the total miR-223 levels are not changed, the percentage of miR-223 associated with Ago2 complexes is increased during ATRA-induced differentiation. F) The resistance of miR-223 in HL60 cell-derived MVs to RNaseA. *, p,0.05; **, p,0.01. doi:10.1371/journal.pone.0046957.gunusual stability of the circulating miRNAs, particularly the miRNAs in cell-derived MVs, provides a solid grounding for the circulating miRNAs to serve as an ideal biomarker for variousdiseases and also as a novel class of signaling molecules in cell-cell communication. Unlike other RNA species, circulating miRNA remains stable in the peripheral blood and culture medium for long periods due toAgo2 Complexes Protect Secreted miRNAsthe significant resistance of the nuclease to degradation. The specific role of Ago2 complexes in the stability of circulating miRNAs has been tested in the present study. Through the disruption of the association of miRNAs, including mi.Otection provided by Ago2 complexes to the various miRNAs in the MVs was different. This finding may raise an important question about the fate of the circulating miRNAs in the cell-derived MVs. Because Ago2 is also a key effector of miRNA function, our results may imply that only the secreted miRNAs that are associated with Ago2 complexes in the cell-derived MVs are stable and have biological function after they enter into the recipient cells, whereas the non-Ago2 complex-bound miRNAs in the MVs may be simply degraded in the recipient cells. Our results further showed that thestability of a circulating miRNA in the cell-derived MVs is positively correlated with the degree of its association with Ago2 complexes. As shown in the in vitro digestion assay with RNase A (Figure 3), Ago2 complex-associated miR-16 was significantly resistant to RNase A compared with free miR-16, which was rapidly degraded by RNase A. These results suggest that certain cell secreted miRNAs are pre-loaded with Ago2 complexes in MVs released by origin cells and can be delivered into recipient cells where they start inhibiting their targets. In other words, the secreted miRNAs in MVs are already functionally equipped with Ago2 and can directly execute their roles in the recipient cells. Therefore, MV-delivery of secreted miRNAs provides a new mechanism for cell-to-cell communication. The Ago2/miRNA complexes are also highly protease-resistant, as miRNA remained stable in the cell lysates for over a week (data not shown). TheAgo2 Complexes Protect Secreted miRNAsFigure 5. Enhancement of both the association of miRNAs with Ago2 complexes in cell-derived MVs and the resistance of miRNAs to RNaseA under various physiological conditions. A) The apoptosis of HeLa cells induced by serum starvation and TNFa treatment for 24 h, respectively. B) Relative levels of total and Ago2 complex-associated miR-16, miR-30a, miR-223 and miR-320b in the MVs derived from HeLa cells with or without apoptotic reagent treatment. Note that, although the total miR-16 levels are not changed, the percentage of miR-16 associated with Ago2 complexes is increased under apoptosis induced by either serum starvation or TNFa treatment. C) The resistance of miR-16 in HeLa cell-derived MVs to RNaseA. D) The differentiation of HL60 cells induced by 20 mM ATRA for 48 h. E) Relative levels of total miR-16, miR-30a, miR-223, miR-320b and the levels of these miRNAs associated with Ago2 complexes in the MVs derived from HL60 cells with or without induced by ATRA. Note that, although the total miR-223 levels are not changed, the percentage of miR-223 associated with Ago2 complexes is increased during ATRA-induced differentiation. F) The resistance of miR-223 in HL60 cell-derived MVs to RNaseA. *, p,0.05; **, p,0.01. doi:10.1371/journal.pone.0046957.gunusual stability of the circulating miRNAs, particularly the miRNAs in cell-derived MVs, provides a solid grounding for the circulating miRNAs to serve as an ideal biomarker for variousdiseases and also as a novel class of signaling molecules in cell-cell communication. Unlike other RNA species, circulating miRNA remains stable in the peripheral blood and culture medium for long periods due toAgo2 Complexes Protect Secreted miRNAsthe significant resistance of the nuclease to degradation. The specific role of Ago2 complexes in the stability of circulating miRNAs has been tested in the present study. Through the disruption of the association of miRNAs, including mi.
