Page 192 - Haematologica Vol. 107 - September 2022

ARTICLE - NOTCH2 in myeloma-derived extracellular vesicles D. Giannandrea et al.
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Figure 6. γ-secretase blockade of NOTCH2 activation inhibits the effect of multiple myeloma cell-released extracellular vesicles on osteoclastogenesis and angiogenesis. (A) Experimental rationale. Multiple myeloma cell-released extracellular vesicles (MM- EV) derived NOTCH2 may increase NOTCH signaling activation on target cells (osteoclast [OCL] progenitors and endothelial cells [EC]) that may be blocked by DAPT administration. (B) Raw267.4 cells induced to differentiate into OCL in the presence of 30 ng/mL RANKL were treated with MM-EV collected from the osteoclastogenic cell line RPMI8226 or the control fresh medium (w/o EV) and in the presence or absence of DAPT to inhibit NOTCH signaling activation. For each condition a negative control cultured in the absence of RANKL was carried out. After 7 days TRAP+ multinucleated cells (≥3 nuclei) were enumerated. The graph shows the mean values of TRAP+ multinucleated cells obtained in the different conditions for RANKL treated cells. Given the large number of conditions, to make the graph simpler and easier to understand, each value was subtracted of the respective control without RANKL (+/- standard error of the mean [SEM]). Statistical analysis was performed by a one-way ANOVA with Tukey post-test; *P<0.05;***P<0.001. (C) Tube formation assay on primary human pulmonary arterial cells (HPAEC) was performed for 13 hours (h) with MM-EV collected from RPMI8226 and OPM2 cells or control fresh medium (w/o EV) in the presence or the absence of DAPT. The graphs show the mean values of areas and nodes enumerated in four quadrant of the well +/- SEM. Stat- istical analysis was performed by ANOVA and Tukey post-test; *P<0.05, **P<0.01. (D) Tube formation assay on HPAEC treated for 13 h with EV collected from the bone marrow of MGUS patients or MM patients in the presence or the absence of DAPT. The graphs show the mean values of areas and nodes enumerated in 4 quadrant of the well (+/- SEM). Statistical analysis was per- formed by ANOVA and Tukey post-test; **P<0.01, ****P<0.0001. The characteristics and number of MM patients are reported in the Online Supplementary Table S1.
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