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. 2020 Mar 20;11:348. doi: 10.3389/fphar.2020.00348

Figure 7.

Figure 7

Muscone inhibits RANKL-induced TRAF6 binding to RANK. (A, B) After induction of M-CSF (20 ng/ml) and RANKL (50 ng/ml) on day 0, BMMs and RAW264.7 cells are divided into groups according to the different times that 40 μM muscone is added (day 1-7 group, day 3-7 group, and day 5-7 group, since muscone is added on day 1, 3, and 5, respectively). On day 7, RANK and TRAF6 mRNA expression in BMMs are detected by RT-PCR for each group. (C, D) RAW264.7 cells are stimulated, with or without RANKL (100 ng/ml) for 20 min, and with or without muscone (40 μM) for 6 h. Cells are then subjected to immunofluorescence staining. The nucleus percentage counting of immunofluorescence staining indicates that muscone significantly downregulated the expression of TRAF6 protein rather than RANK. (E) To verify the result, coimmunoprecipitation shows that TRAF6 protein levels of RAW264 cells treated with vehicle or muscone (40 μM) with or without RANKL, measured by Western blot analysis. Input: TRAF6 and IB: RANK was used as loading controls. (F) Schematic diagram of the mechanism by which muscone ameliorates RANKL-induced osteoclastogenesis via blocking the binding of TRAF6 to RANK. *P < 0.05 vs. the control group, #P < 0.05 vs. the RANKL-induced group.