Research Paper Volume 15, Issue 16 pp 8220—8236

Figure 4. VCP20 inhibits osteoclast differentiation via downregulating NF-κB P65. (A) TRAP staining showed that VCP20 inhibited osteoclast differentiation in RAW 264.7 cells treated with RANKL and M-CSF. Scale bar: 250 μm. (B) Quantitative analysis of multinucleated osteoclasts. (C) The biological characteristics of exosomes were detected by transmission electron microscopy. Scale bar: 100 nm. (D) Detection of VCP, Alix and β-actin in ARP1 WT cells and exosomes from ARP1 WT cells by WB analysis. (E) Exosomes from ARP1 WT cells rescued cell proliferation inhibited by VCP20 in MM cells (1×10⁶ MM cells were treated with 20 μg exosomes or not for 48 h). (F) TRAP staining revealed that exosomes from ARP1 WT cells induced osteoclast differentiation. Scale bar: 250 μm. (G) Quantitative analysis of multinucleated osteoclasts. (H) WB assay confirmed that exosomes from ARP1 WT cells increased the expressions of P-P65 and NFATC1 that were downregulated by VCP20 in RAW264.7 cells. (I) RT-PCR assay showed that VCP20 decreased the levels of osteoclast differentiation related markers, and this effect was partially compensated by exosomes from ARP1 WT cells. VCP20: 100 nM; exosomes: 2 μg/mL each well. Data are presented as the mean ± SD; *p < 0.05; **p < 0.01; ***p < 0.001.