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. 2020 Dec 23;8:594785. doi: 10.3389/fcell.2020.594785

FIGURE 3.

FIGURE 3

Exosomal circHmbox1 from osteoclasts treated with TNF-α inhibits osteoblast differentiation. (A) An electron microscopy image of exosomes. Scale bar = 0.5 μm. (B) The expressions of CD63, CD81 and HSP70 in cells lysates and exosomes secreted by RANKL-induced RAW 264.7 cells were analyzed by western blot. (C) The expressions of circHmbox1 were analyzed by qRT-PCR in osteoblasts incubated with exosomes from osteoclasts induced with RANKL and/or TNF-α. n = 4, **P < 0.01. (D) Confocal microscopy images of colocalization of exosomes from RANKL-induced RAW 264.7 cells with osteoblasts. Exosomes were labeled by DiO (green), F-actin were stained with rhodamine-phalloidin (red) and cell nuclei were stained with Hoechst 33342 (blue). Scale bars, 25 μm. (E) The expressions of circHmbox1 were analyzed by qRT-PCR in osteoblasts co-cultured with osteoclasts induced with RANKL and/or TNF-α. n = 4, **P < 0.01. (F) Osteoblasts adding exosomes from osteoclasts increasing or silencing circHmbox1 were cultured in osteogenic differentiation medium for 7 days. QRT-PCR was applied to test the expressions of Osterix and Runx2. n = 4, **P < 0.01. (G) Osteoblasts adding exosomes from osteoclasts increasing or silencing circHmbox1 were cultured in osteogenic differentiation medium for 7 days, followed by ALP staining. n = 4. (H) The expressions of CD81 and TSG101 in cells lysates and exosomes secreted by osteoclasts treated with GW4869 were analyzed by western blot. n = 3. (I) The expressions of Runx2 and Osterix were analyzed by qRT-PCR in osteoblasts co-cultured with osteoclasts treated with GW4869 in the presence of RANKL and TNF-α. n = 3, **P < 0.01. (J) Osteoblasts were co-cultured with osteoclasts treated with GW4869 in the presence of RANKL and TNF-α, followed by ALP staining. n = 4. (K–M) Osteoblasts adding exosomes from osteoclasts increasing or silencing circHmbox1 were cultured in osteogenic differentiation medium for 7 days in presence of TNF-α. QRT-PCR was applied to test the expressions of Runx2 (K) and Osterix (L). n = 4, **P < 0.01. (M) Osteoblasts adding exosomes from osteoclasts increasing or silencing circHmbox1 were cultured in osteogenic differentiation medium for 7 days in presence of TNF-α, followed by ALP staining. n = 4.