C57b6 l mice

Osteoclast differentiation was performed as previously described [18 (link)]. Bone marrow cells obtained from 6- to 8-week-old C57B6/L mice (Dae Han Bio Link, Chungbuk, Korea) were incubated in α-minimal essential medium (α-MEM) containing 10% fetal bovine serum (FBS). After 24 h, non-adherent cells were centrifuged on a Histopaque density gradient (Sigma-Aldrich, St. Louis, MO, USA) and cultured in α-MEM supplemented with 10% FBS and M-CSF (30 ng/mL) for 3 days to obtain bone marrow macrophages (BMMs). BMMs were cultured with RANKL (20 ng/mL) and M-CSF (10 ng/mL) in the absence or presence of 1 μM or 5 μM OCLI-023 for 4 days. Then, the cells were stained with a tartrate-resistant acid phosphatase (TRAP)-staining solution prepared following the manufacturer’s instructions (Sigma-Aldrich). TRAP-positive multinucleated cells (MNCs), having three or more nuclei, were counted as osteoclast-like cells.

Osteoclast differentiation was induced as previously described [17 (link)]. Bone marrow cells collected from 6–8 week-old C57B6/L mice (Dae Han Bio Link, Chungbuk, Korea) were cultured in α-minimal essential medium (α-MEM) supplemented with 10% fetal bovine serum (FBS). Next day, non-adherent cells were collected, centrifuged in Histopaque density gradient (Sigma—Aldrich, St. Louis, MO), and incubated in α-MEM containing 10% FBS and M-CSF (30 ng/mL) for 3 days. Attached cells were considered to be BMMs. In order to induce osteoclast differentiation, BMMs were cultured in α-MEM supplemented with 20 ng/mL RANKL and 10 ng/mL M-CSF in the absence or presence of 1 μM or 5 μM KP-A159. Osteoclast formation was investigated by TRAP staining following the manufacturer’s instructions (Sigma—Aldrich). TRAP-positive multinucleated cells (MNCs) containing ≥3 nuclei were calculated as osteoclast-like cells.