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. 2015 Apr 12;6(15):13772–13789. doi: 10.18632/oncotarget.3830

Figure 8. Exosomes in plasma of patients with multiple myeloma promote osteoclasts differentiation from Raw264.7 precursors and regulate bone resorption activity of osteoclasts.

Figure 8

A. Raw264.7 cells were incubated with 25ng\ml of hrRANKL (positive control) or with 25 μg/ml of exosomes isolated from plasma of two patients with MM for six days, and then stained for TRAP expression. TRAP positive cells (i.e., those containing three nuclei) were photographed and counted. Right panel shows representative pictures of Raw264.7 cells stained for TRAP (original magnification 20x). Data presented are the mean of three separate experiments. Sidak test:, EXO-Patient-1/-2 vs Untreated (*p < 0.05); °,hrRANKL vs Untreated (p < 0.05); B. Murine Metalloproteinase-9 (mMMP9) protein levels in the conditioned medium derived from Raw264.7 cells untreated or treated for six days with 25ng\ml of hrRANKL (positive control) or with 25 μg/ml of exosomes isolated from plasma of two patients with MM were determined by enzyme-linked immunoadsorbent assay (ELISA). Data presented are the mean of three separate experiments. Sidak test: *,EXO-Patient-1/-2 vs Untreated (*p < 0.05); °,hrRANKL vs Untreated (°p < 0.05); C. Bone resorption ability of Raw264.7 cells treated for six days with 25ng\ml of hrRANKL (positive control) or with 25 μg/ml of exosomes isolated from plasma of two patients with MM, was evaluated by resorption pit assay on dentine discs. Data relative to the resorbed areas by mature osteoclasts are represented as fold increase of Raw264.7 cells untreated (black column, 1 arbitrary unit). Data presented are the mean of three separate experiments. Sidak test: *,EXO-Patient-1/-2 vs Untreated (*p < 0.05); °,hrRANKL vs Untreated (p < 0.05); Right panel shows representative pictures of lacunae (dark areas) formed by cells treated with rhRANKL or exosomes from MM patients (original magnification 20x).