Figure 6.

Endocytosis-related proteins are involved in the uptake of BMSC-derived sEVs by MM cells. RPMI 8226 and U266 cells were infected with lentivirus expressing shRNA against key endocytic proteins, including (A) CAV-1, (C) CLTC, (E) DNM2, and (G) Flot1 or negative control shRNA (shNC) and the expression of these proteins was determined using western blot. The pixel density of endocytosis-related proteins was quantified and normalized to GAPDH. RPMI 8226 and U266 cells infected with lentivirus expressing shRNA against key endocytic proteins, including (B) CAV-1, (D) CLTC, (F) DNM2, and (H) Flot1, or shNC were cultured with 50 μg/mL DID-labeled sEVs for 4 h and the mean fluorescence intensity of DID in these cells was determined using flow cytometry. Student's t-test was used for comparing two groups. (I-K) RPMI 8226 cells were infected with lentivirus expressing shRNA against DNM2 or CLTC or negative control shRNA (shNC) for 24 h and treated with or without bortezomib in the presence or absence of 100 μg/mL sEVs for another 48 h. (I) Their cell viability was measured using a luminescent cell viability assay. One representative result in triplicate of three experiments was presented by histograms. (J) Apoptotic cells were determined using 7-AAD and Annexin-V staining and flow cytometry. The proportions of live, early apoptotic or late apoptotic and dead cells were analyzed and presented by histograms. (K) Representative flow cytometry plots are shown. One-way ANOVA followed by multiple compressions was used for comparing multiple groups. Error bar, mean ± SD. **P < 0.01, ***P < 0.001, ****P < 0.0001.