Fig. 3: Intake of T-ALL and B-ALL EVs by lipid raft-enriched HSPC.

A Procedure followed to identify populations of murine Lin⁻ cells that take in fluorescent ALL EVs. Murine Sca1⁺ cells were purified with magnetic beads and stained with CTB prior to incubation with fluorescent ALL EVs (HSP70-ATTO488). B Flow cytometry on different populations showing that LSK and HSC (SLAM cells) express high levels of lipid rafts (CTB-AF555) as well as intake of ALL EVs (ATTO488 EVs). Sca1⁺ cells show intermediate levels in expression of lipid rafts and ALL EV intake, while Sca1⁻ cells are not positive for lipid rafts expression or for intake of ALL EVs. Representative of two independent experiments. C Flow cytometry on HSC showing that when Sca1⁺ cells are pretreated for 2 h with Methyl-β-cyclodextrine (Mβc), this affects the lipid rafts staining (CTB-AF555) as well as the intake of ALL EVs (ATTO488 EVs). D Microscopy on murine Sca1⁺ cells showing that the population of primitive hematopoietic cells which take in fluorescent EVs (ATTO488 EVs) correspond to cells also displaying lipid raft staining with the cholera toxin subunit B (CTB-AF555). Magnification ×40, scale bar represents 5 µm. Representative of two independent experiments. E Single cell sorting on positive cells showing colocalization score (R) between EV intake and lipid rafts. Data shows box-and-whisker plots (n = 30 cells for each conditions), presented as medians (central line), first and third quartiles (bottom and top of boxes, respectively), and whiskers (extreme values). Fluorescent optical sections of cells, magnification ×63, scale bar represents 5 µm.