Isolation of bone marrow precursors and the in vitro osteoclastogenesis experiment were performed as described previously (Choi et al., 2013 ). In brief, bone marrow cells isolated from femurs of 4–6 week-old C57BL/6 male mice were cultured in the presence of M-CSF (20 ng/ml, R&D Systems) for 3 days. After washing out the non-adherent cells, the adherent cells were used as BMMs. For osteoclast formation, the isolated preosteoclasts were further cultured in the presence of 200 ng/ml of RANKL (Huh et al., 2016 (link)), 30 ng/ml of M-CSF and/or BAY 60-6583 (Tocris). After 3 days, the cells were fixed and stained for tartrate-resistant acid phosphatase (TRAP) using a TRAP staining kit (Sigma). The cells were observed using a Zeiss Axiovert 200 microscope and images were obtained with an AxioCam HR (Carl Zeiss) equipped with Axio Vision 3.1 software (Carl Zeiss). TRAP-positive multinucleated cells (TRAP+ MNCs) larger than 100 μm in diameter containing more than 20 nuclei and TRAP+ mononuclear cells were counted, and the number was presented as relative percentage (%).