Trap staining solution

The distilled water extract of DM was purchased from the Korean Plant Extract Bank (Daejeon, Korea). The TRAP staining solution was purchased from Sigma Aldrich (St. Louis, MO, USA), and the XTT assay kit was obtained from Roche (Indianapolis, IN, USA). The α-minimum essential medium (α-MEM), fetal bovine serum (FBS), and penicillin-streptomycin were purchased from Gibco-BRL (Grand Island, NY, USA); soluble human recombinant M-CSF and RANKL were purchased from Peprotech (London, UK). Specific primary antibodies against phospho-p38 (#9211), p38 (#9212), phospho-JNK (#9251), JNK (#9252), phospho-Akt (#9271), Akt (#9272), and phospho-IκB (#2859) were purchased from Cell Signaling Technology (Beverly, MA, USA), while phosphor-Btk (GTX61791) was purchased from GeneTex (Irvine, CA, USA) and β-actin (A5441; housekeeping gene) was obtained from Sigma Aldrich. Specific antibodies against phospho-PLCγ2 (sc-101785), PLCγ2 (sc-5283), IκB (sc-371), and c-Fos (sc-7202), and NFATc1 (sc-7294) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Alpha-minimum essential medium (MEM), fetal bovine serum, and penicillin were purchased from Gibco BRL. Recombinant mouse M-CSF was purchased from R&D Systems. Recombinant Murine sRANK Ligand was purchased from Peprotech. TRAP staining solution was obtained from Sigma-Aldrich. The Cell Counting Kit-8 (CCK-8) was obtained from Dojindo Molecular Technology. The Click-iT® EdU Imaging Kits (C10337) were purchased from Invitrogen Life Technologies.

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.

The mouse femurs were fixed and decalcified, and then embedded in paraffin. Sections of 4.5 μm were prepared. H&E staining (Solarbio, Beijing, China) was performed as the manufacturer’s instruction. The stained sections were observed using an inverted microscope (IX81, Olympus).
TRAP staining was performed according to the procedure described previously (Liu et al., 2016 (link)). TRAP staining solution (Sigma) was added to cover the tissue sections. After incubating at 37°C for 40 min, the sections were stained with 0.1% methyl green solution (Sigma) for 10 s. The stained sections were observed using an inverted microscope (IX81, Olympus).

Primary mouse osteoclasts and RAW264.7 cells were seeded in 24-well plates and differentiated for seven days. After induction of differentiation, cells were washed with phosphate buffered saline (PBS) and fixed in paraformaldehyde 4% in PBS for 10 min, following by incubating for 20 min at 37 °C in the TRAP staining solution (Sigma-Aldrich) according to the manufacturer’s instructions.