Fig. 1.

CD47 differentially regulates the abundance of specific miRNAs in cells and EVs. a Hierarchical clustering of differentially expressed miRNAs in EVs (> twofold, p val < 0.05) with unsupervised clustering of their parental cell data based on microarray analyses of WT and CD47 deficient Jurkat T cells and their released EVs (N = 3) b Hierarchical clustering of differentially expressed cellular miRNAs with unsupervised EV data. c Principal component analysis of miRNA data for WT and CD47⁻ Jurkat T cells and EVs released by these cells. d Venn diagram summarizing RNAseq analysis of differentially expressed miRNAs (p < 0.05) comparing WT and CD47⁻ cells (Blue) and EVs (Red) (Data S2A, p < 0.05). The left pie chart depicts the percentages of miRNAs up- or down-regulated only in CD47⁻ versus WT EVs. The right pie chart presents the percentages of miRNAs up (orange) or down regulated (gray) in CD47⁻ EVs and in cells and miRNAs showing opposite CD47-dependent regulation in cells versus EVs (dark blue). e, f Venn diagram presenting numbers of precursor e and mature miRNAs f differentially expressed in WT vs. CD47⁻ cells (blue) and WT vs CD47⁻ EVs (red) (Data S2A, p < 0.05). g Confirmation using real time PCR of representative differentially regulated miRNAs between WT and CD47⁻ cells and their EVs (Data S1C). h WT cells were transfected with a CD47 guide CRISPR/Cas9and grown in complete medium for 24 h. Transfected cells were transferred into HITES serum free medium for 24 h. EVs were isolated using the Exo-quick Kit, and miRNA levels of miR-31, miR-107 and miR-103 was analyzed using real-time PCR. Significance is indicated for P-values ≤ 0.05 for comparisons of Sample and Columns (*) or Sample, Columns and Interaction (**) as detailed in the Statistical Analysis method section. i CD47⁻ cells were transfected for 48 h with control or CD47 expression plasmids. Total RNA from cells and EVs was analyzed for expression of the indicated miRNAs. The level was normalized to EVs extracted from WT using U6 as control. Significance was determined by two-sample t-test assuming equal variances (P-value ≤ 0.05). j WT and CD47⁻ T Cells were transferred into serum free HITES medium for 24 h. EVs were isolated using ultracentrifugation basic protocol 1 and k size exclusion chromatography purification using Exo-guidance systems, and real time PCR was performed. l RNA was extracted from CD3⁺ T cells isolated from WT and Cd47^−/− mice, and miRNA level was analyzed using real time PCR. Significance is indicated for P-values ≤ 0.05 with two-sample t-test: assuming equal variances