And 3B, Ago2 complexes strongly protect miR-16 against RNaseA degradation in a timeand dose-dependent fashion, and the protection by the Ago2 complexes can be completely abolished by PK treatment. Recently, a small molecule named trypaflavine (TPF) has been discovered to block the loading of miRNAs into Ago2 complexes, possibly through disruption of the protein-protein association between TRBP and Ago2 [30]. We tested whether TPF treatment can decrease the stability of miRNAs, including miR-16, miR-30a, ��-Sitosterol ��-D-glucoside chemical information miR-223 and miR-320b, in secreted MVs by decreasing the miRNA-Ago2 association. In this experiment, HeLa cells were treated with or without 8 mM TPF for two days. The MVs were collected from the culture media and then used for an Ago2 pulldown assay. As shown in SR3029 Figure 4A, we found no change in the total amount of each miRNA in the MVs, but the percentage of Ago2 complex-associated miRNAs was significantly reduced. This decrease of not the total miRNA level 22948146 but the level of miRNA associated with Ago2 was also observed in HeLa cells treated with TPF (Figure S2B). Interestingly, the level of Ago2 in HeLa cells was not altered by TPF treatment (Figure S2A). As expected, the stability of miR-16 in the MVs derived from the TPF-treated HeLa cells was significantly lower than that of non-treated MVs (Figure 4B). It has been shown that miR-16 [31] and miR-223 [32,33] are linked to cellular apoptosis and differentiation process, respectively. Our previous study also showed that the intracellular distribution of miRNAs may be related to certain cellular 
functional states [24]. To study whether the association of MVencapsulated miRNAs with Ago2 complexes and their resistance to RNaseA degradation is dynamically regulated by cellular biological function, we assessed the relationship between the association of Ago2 complexes with miR-16 or miR-223 and the resistance of these miRNAs to RNaseA under cell apoptotic or differentiation conditions. In these experiments, HeLa cells were treated with tumor necrosis factor a (TNFa) or serum-depleted cultured medium to induce apoptosis, while promyelocytic HL60 cells were treated with ATRA to induce cell differentiation [34]. The percentage of apoptotic HeLa cells was increased under both serum deprivation and TNFa treatment (Figure 5A). The MVs released by the HeLa cells were then collected from the culture medium for stability analysis. As shown in Figure 5B, under the early cell apoptotic conditions induced by serum depletion or TNFa, the percentage of miR-16 associated with Ago2 complexes in the MVs was markedly increased, although the total amount of miR-16 was not changed. A similar elevation of Ago2 complexassociated miR-16 but not total miR-16 was also observed in apoptotic HeLa cells (Figure S3A, lower panel). We also tested the total amount of cellular Ago2 under normal and apoptotic conditions and found no enhancement of the Ago2 expression level by apoptosis (Figure S3A, upper 12926553 panel). As expected, with the percentage of Ago2-associated miR-16 being increased, the resistance of the miR-16 in the MVs to RNaseA was significantly enhanced (Figure 5C). TNFa treatment of HeLa cells also caused alteration of many miRNAs at cellular level. For example, the level of miR-483-5p in HeLa cells was upregulated by TNFa treatment (Figure S3, lower panel). We also tested the level of miR-483-5p and its association with Ago2 in MVs, and the data 
indicated that the levels of miR-483-5p associated with or without Ago.And 3B, Ago2 complexes strongly protect miR-16 against RNaseA degradation in a timeand dose-dependent fashion, and the protection by the Ago2 complexes can be completely abolished by PK treatment. Recently, a small molecule named trypaflavine (TPF) has been discovered to block the loading of miRNAs into Ago2 complexes, possibly through disruption of the protein-protein association between TRBP and Ago2 [30]. We tested whether TPF treatment can decrease the stability of miRNAs, including miR-16, miR-30a, miR-223 and miR-320b, in secreted MVs by decreasing the miRNA-Ago2 association. In this experiment, HeLa cells were treated with or without 8 mM TPF for two days. The MVs were collected from the culture media and then used for an Ago2 pulldown assay. As shown in Figure 4A, we found no change in the total amount of each miRNA in the MVs, but the percentage of Ago2 complex-associated miRNAs was significantly reduced. This decrease of not the total miRNA level 22948146 but the level of miRNA associated with Ago2 was also observed in HeLa cells treated with TPF (Figure S2B). Interestingly, the level of Ago2 in HeLa cells was not altered by TPF treatment (Figure S2A). As expected, the stability of miR-16 in the MVs derived from the TPF-treated HeLa cells was significantly lower than that of non-treated MVs (Figure 4B). It has been shown that miR-16 [31] and miR-223 [32,33] are linked to cellular apoptosis and differentiation process, respectively. Our previous study also showed that the intracellular distribution of miRNAs may be related to certain cellular functional states [24]. To study whether the association of MVencapsulated miRNAs with Ago2 complexes and their resistance to RNaseA degradation is dynamically regulated by cellular biological function, we assessed the relationship between the association of Ago2 complexes with miR-16 or miR-223 and the resistance of these miRNAs to RNaseA under cell apoptotic or differentiation conditions. In these experiments, HeLa cells were treated with tumor necrosis factor a (TNFa) or serum-depleted cultured medium to induce apoptosis, while promyelocytic HL60 cells were treated with ATRA to induce cell differentiation [34]. The percentage of apoptotic HeLa cells was increased under both serum deprivation and TNFa treatment (Figure 5A). The MVs released by the HeLa cells were then collected from the culture medium for stability analysis. As shown in Figure 5B, under the early cell apoptotic conditions induced by serum depletion or TNFa, the percentage of miR-16 associated with Ago2 complexes in the MVs was markedly increased, although the total amount of miR-16 was not changed. A similar elevation of Ago2 complexassociated miR-16 but not total miR-16 was also observed in apoptotic HeLa cells (Figure S3A, lower panel). We also tested the total amount of cellular Ago2 under normal and apoptotic conditions and found no enhancement of the Ago2 expression level by apoptosis (Figure S3A, upper 12926553 panel). As expected, with the percentage of Ago2-associated miR-16 being increased, the resistance of the miR-16 in the MVs to RNaseA was significantly enhanced (Figure 5C). TNFa treatment of HeLa cells also caused alteration of many miRNAs at cellular level. For example, the level of miR-483-5p in HeLa cells was upregulated by TNFa treatment (Figure S3, lower panel). We also tested the level of miR-483-5p and its association with Ago2 in MVs, and the data indicated that the levels of miR-483-5p associated with or without Ago.