Extended Data Fig. 8: Additional information about mutations in miR-34c.

a) Table depicting the name and sequence of the different EXOmotifs introduced in miR-34c-5p. Bold underlying text in the sequence indicates changed nucleotides in the guide strand of the miRNA. Nucleotides in the passenger strand were also modified accordingly to maintain miRNA structure. b) Predicted structure for the hairpin miRNA for each of the constructs shown in a. Red means high probability of pairing while blue indicates low probability calculated by RNAfold WebServer software. Arrows indicate the ___location of the mutated nucleotides. c) Normalized expression for miR-34c-5p wild-type in cells and sEV for each cell type. Average Ct from the whole miRNA profile was used for normalization for each sample. *P≤0.05 (Limma t-test). d) To visualize the changes in the sEV and cell content of each miRNA construct before sEV enrichment calculation, normalized expression of miR-34c wild-type (WT) or its EXOmotif-containing versions miR-34-UGUGU, miR-34-CAUG and miR-34-CGGGAG are displayed for the cells overexpressing each of the miR-34c versions. miR-138-5p was used to normalize expression in sEV versus cells as we previously showed that the levels of this miRNA are equivalent in both compartments.*P≤0.05 (Mann-Whitney U test comparing sEV and cell expression for the same miRNA version, i.e. red versus blue bars). e) Absolute copy number of the miRNAs displayed in the x-axis overexpressed in brown adipocytes were quantified in sEV isolated by ultracentrifugation followed by size exclusion chromatography and normalized by the miRNA copy number in all producing cells for each sample. f) sEV enrichment calculated as the ratio of sEV expression divided by cellular expression for each of the constructs expressed in and secreted from AML12 hepatocytes. g) sEV enrichment calculated as the ratio of sEV expression divided by cellular expression for each of the constructs expressed in and secreted from SVEC endothelial cells. In both panels f, g, the dashed line separates preferential sEV enrichment (above line) versus preferential cellular enrichment (below line). Expression was normalized to the expression of miR-501-5p, which is to be equally abundant in sEV and cells for each cell type. Data are expressed as mean ± SEM. n=3, *P≤0.05, Kruskal-Wallis followed by Mann-Whitney U tests.