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. 2021 Mar 13;11(11):5491–5510. doi: 10.7150/thno.55041

Figure 6.

Figure 6

EVs loaded miR-143/145 from BMSCs activates osteoclast function. (A) A representative image of EVs from BMSCs by transmission electron microscopy (TEM). Scale bars: 100 nm. (B) NTA of EVs for calculating the size distribution. The left Y-axis indicated particle number concentration and X-axis indicated particle size. (C) Cd63, Cd9, and Calnexin from EVs and BMSCs were detected by Western blot. (D) Fluorescent microscopy and TRAP staining analysis revealing DiI-labeled EVs injected into bone marrow. Red arrow head indicated DiI-labeled EVs. Scale bars: 50 μm. (E) miR-143/145 loaded in EVs from Sham and OVX-induced BMSCs were examined by qRT-PCR. (F) EVs from OVX BMSCs, miR-143/145 mimics treated BMSCs or miR-143/145-/- BMSCs were co-cultured with osteoclasts and visualized by TRAP staining. Scale bars: 100 μm. Quantitative measurements of (G, H) TRAP positive staining cells, (I, J) F-actin ring staining cells, and (K, L) bone resorption area in miR-143/145 inhibitor or mimics treated osteoclasts. (M) Western blot showing the Cd226 and Srgap2 protein levels in EVs treated osteoclasts. (N) Western blot showing the Cd226 and Srgap2 expression in miR-143/145 inhibitor or mimics treated osteoclasts. (O, P) WT and mutation of Cd226 or Srgap2 vectors were subjected to dual-luciferase reporter assays. (Q) A schematic working model for miR-143/145 shuttling from BMSCs to osteoclasts through EVs. Results are presented as the mean ± S.D. *p < 0.05; **p < 0.01; #p > 0.05 by Student's t test and one-way ANOVA.