Page 189 - Haematologica Vol. 107 - September 2022

ARTICLE - NOTCH2 in myeloma-derived extracellular vesicles D. Giannandrea et al.
mentary Figure S5 shows that NOTCH2 KD did not affect the expression of NOTCH1, 3 and 4 in protein extracts from cells and MM-EV. The outcome of NOTCH2 KD on EV size and concentration evaluated by NTA showed no sig- nificant effect on MM-EV size and concentration (Figure 4B).
In order to assess if the NOTCH2 protein carried by MM- EV is functionally active and is able to trigger NOTCH sig- naling in receiving cells, we tested the effect of EV isolated from HMCLN2KD (MM-EVN2KD) or HMCLSCR (MM-EVSCR) through a NOTCH responsive luciferase reporter assay. This assay was carried out in HeLa cells, which is a highly-
transfectable cell line characterized by a low level of NOTCH signaling activation (not shown). Figure 4C shows that MM-EVSCR can activate the NOTCH signaling pathway in the receiving HeLa cells, while this ability is significantly reduced for MM-EVN2KD.
The ability of MM-EV to activate NOTCH signaling was also validated in an in vivo NOTCH reporter zebrafish embryo obtained by crossing Tg(T2KTp1bglob:hmgb1-mCherry) with Tg(fli1a:EGFP) that carry EGFP+ endothelial cells (green) and express the mCherry protein (red) under the control of a NOTCH-responsive element. EV isolated from RPMI8226 cells were injected in the duct of Cuvier of 48 hpf
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