Fig 2: miRNA hierarchical clustering analysis. Forty out of the 84 measured miRNAs with the highest coefficient of variation were chosen for the analysis (selected miRNAs names are indicated on the Y‐axis in the left). The colored miRNAs names are RBC‐ (red) and WBC‐ (grey) associated miRNAs. The expression level of these 40 miRNAs is represented as a colored heat‐map (Y‐axis bar in the right). The correlation of miRNAs expression was used as a parameter for clustering distance. Three major clusters were formed: Ago1 + Ago2 RIP miRNAs, whole serum and exosomal miRNA and Ago1 associated miRNAs (indicated on X‐axis top). The numbers on the X‐axis in the lower bar indicate donors (n = 3)

Fig 3: (A,B) Values of Ago1‐HF score, calculated as a sum of miR‐222‐3p, let‐7a‐5p, miR‐497‐5p and miR‐21‐5p relative expression values, in Ago1 RIP (A) and whole serum (B). (C,D) ROC curve for HF score of healthy individuals (n = 10) vs HF patients (n = 8) in Ago1 RIP (C) and whole serum (D)

Fig 4: miRNAs differentially associated with Ago1 vs Ago2 proteins in serum and plasma samples. Ago1‐ and Ago2‐assosiated miRNAs detected in serum and plasma samples were selected based on the following criteria: median fold change > 2 and P < .05 between Ago1 and Ago2 serum samples, and Ct < 37.5 (for both serum and plasma samples of all 3 donors). The colored miRNAs are RBC (red) and WBC (grey) associated miRNAs. Samples of three healthy donors were used for the analysis

Fig 5: The co-chaperone Fkbp5 interacts with Ago2 in mouse ESCs. (A) Cell extracts from KH2-Flag-Ago2 ESCs grown in dox-containing media were subjected to Flag-affinity purification. The affinity eluate was analyzed by SDS-PAGE followed by immunoblotting (IB) using α-Flag antibody, silver staining, and colloidal blue staining followed by mass spectrometry analysis. Arrowhead depicts Ago2, and asterisks depict common contaminants. (B) Partial list of proteins identified by mass spectrometry. (C) Schematic representation of Fkbp5 domains (FK indicates FKBP12-like domain; TPR, tetratricopeptide repeat). (D) Cell extracts from KH2-Flag-Ago2 and control KH2-Flag-Lin28 grown in dox-containing media were subjected to Flag-immunoprecipitation (IP). Associated proteins were detected by Western blotting using α-HSP90 and α-FKBP5 antibodies. Asterisk depicts unspecific protein detected by the α-FKBP5 antibody. (E) Cell extracts from V6.5 ESCs transfected with pFlag-Ago1, pFlag-Ago2, or pFlag-Lin28 expressing constructs or empty vector (pFlag-CMV2), were subjected to Flag IP. Associated proteins were detected by Western blotting using α-HSP90 and α-FKBP5 antibodies. (F) Cell extracts from KH2-Flag-Ago2 cells grown in dox-containing media were subjected to Flag-affinity purification. Where indicated, extracts were treated overnight with RNase A (20 µg/mL). Associated proteins were detected using the indicated antibodies. Asterisk depicts unspecific protein detected by the α-FKBP5 antibody. (G) FK506 blocks the interaction between Ago2 and Fkbp5. KH2-Flag-Ago2 cells were treated with 1 μg/mL dox and (10 µM) FK506 or DMSO for 24 h. Cell extracts were subsequently subjected to Flag-affinity purification. Because the overall levels of Flag-Ago2 are more abundant in extracts from DMSO-treated cells compared with extracts from FK506-treated cells, Flag-IP eluates were diluted by a factor of eight (1:8) for DMSO sample, while Flag-IP eluates from FK506-treated cells were kept undiluted. The resulting Flag IPs were then assessed for associated Fkbp5 by SDS-PAGE and immunoblotting using an α-FKBP5 antibody. An asterisk depicts unspecific protein detected by the α-FKBP5 antibody.