Figure 3.

Free light chains and GAGs mediate MM EVs intracellular uptake. (A) Fluorescent microscopy analysis of HVECs incubated with MGUS and MM PKH67-labeled EVs (200 μg of proteins) for 4 h at 37°C. Before cell treatment, MM EVs were incubated with sheep anti-FLCs (MM + sh anti FLC), mouse anti-CD63 (MM + sh anti-CD63) antibody or with heparin 100 ng/ml (MM + heparin). (B) Quantitative data from similar experiments analyzed by flow cytometry. HVECs were labeled with CD31-APC (endothelial marker) and EVs were labeled with PKH67-FITC. (C) PKH67 fluorescence intensity measurement of internalized MM EVs incubated with sheep anti-FLCs, mouse anti-CD63 antibody or with heparin 100 ng/ml (MM + heparin) (100 cells each experimental point) using ImageJ program. Significant differences were determined with Student’s t-test. Values were shown as mean values ± SEM of at least three experiments. *p < 0.05, **p < 0.01, ***p < 0.001. (D) HVEC and H9C2 cells in serum-free medium were incubated for 24, 48, and 72 h with 50 μg of MM 5 and MM 6 EVs pre-treated or not with a polyclonal anti-FLCs antibody. Cell proliferation rate (growth factor) was assessed using crystal violet. Mean values ± SEM for three independent experiments are shown. *p < 0.05, **p < 0.01, ***p < 0.001.