Fig. 1

EV-miRNAs characterization and function in T-ALL model. A ddPCR analysis of validated NOTCH1 target genes mRNA expression in CUTLL1-dnMAM vs. CUTLL1-CTRL cells. Y-axes, mRNA levels of NOTCH1 target genes normalized to B2M expression. X-axes, gene symbols. Significance analysis was performed by one-sample t-test. B Immunoblot analysis of HES1, HES4 and c-MYC proteins in CUTLL1-CTRL and CUTLL1-dnMAM cells. C Nanoparticle-tracking-analysis of the size distribution and concentration of sEV released by CUTLL1-CTRL and CUTLL1-dnMAM cells. Inset plots, Transmission Electron Microscope (TEM) images showing particles in sEV samples from CUTLL1-CTRL and CUTLL1-dnMAM cells. Scale bar = 100 nm. On the right, box plots of differential concentration of sEV in CUTLL1-CTRL and CUTLL1-dnMAM cells. Significance analysis was performed by Student t-test. D Immunoblot analysis of sEV markers (CD81, Syntetin1 and CD63) in CUTLL1-CTRL and CUTLL1-dnMAM cells. E Venn diagram of EV-miRNAs detected in CUTLL1-CTRL and CUTLL1-dnMAM cells (CELL) or in their released small extracellular-vesicles (sEV). Significance analysis was performed by Fisher’s exact test. F Hierarchical clustering analysis of miRNAs detected (N = 318) in CUTLL1-CTRL (Ctrl) and CUTLL1-dnMAM (dnM) cells (CELL) and/or in their released small extracellular-vesicles (sEV). On the right, most abundant miRNAs in sEV were also indicated; in bold, members of the miR-17–92 cluster. G On top, violin plots of differential expression (dnMAM vs. CTRL) of the 73 commonly detected miRNAs in CUTTL1 cells and in sEV. Bottom, bar plots of differential expression (dnMAM vs. CTRL) of the miR-17–92 cluster and paralogues. Colors are as per the legend. Significance analysis was performed by Mann–Whitney U-test. H qRT-PCR analysis of miR-17–92 cluster overexpressing CUTLL1-dnMAM cells vs. control (Empty-V) CUTLL1-dnMAM cells. Significance analysis was performed by one-sample t-test. I ddPCR analysis of miR-17–92 cluster in sEV purified from miR-17–92 cluster overexpressing vs. control (Empty-V) CUTLL1-dnMAM cells. Bubble size represents the average expression of miRNAs (copies/20µL). Colours are as per the legend. J Flow cytometry analysis of CUTTL1 (CTRL) and CUTTL1-dnMAM cells (dnMAM) conditioned with PKH26-labelled miR-17–92-enriched sEV (EV_miR-17–92) or PKH26-labelled Empty-Vector sEV (EV_Empty-V) derived from miR-17–92 overexpressing CUTTL1 cells or from CUTLL1 cells transfected with an empty vector, respectively. MFI, mean fluorescence intensity. Percentages of cells which internalized exogenous PKH26-sEV (Cells EV-pos) are also shown. K Viability of CUTTL1 (CTRL) and CUTTL1-dnMAM cells (dnMAM). Briefly, transduced GFP positive cells were FACS sorted and in vitro grown together with miR-17–92-enriched sEV (EV_miR-17–92) or sEV (EV_Empty-V) cultured for two days. GFP + alive cells were measured by flow cytometry for DAPI (4′,6-diamidino-2-phenylindole) exclusion and counted by relating the cell numbers to internal fluorescent bead events (see also methods). The graph reports the result of two independent experiments. Significance analysis was performed by Student’s t-test