Trap stain kit

Recombinant Mouse RANKL and Recombinant Mouse M-CSF were purchased from R&D Systems (Minneapolis, MN, USA). Antibodies against AIF, cytochrome c, cleaved caspase-3, Bax, Bcl-2 and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Osteo Assay Surface for Bone Resorption was purchased from Corning (Corning, NY, USA). Bovine cortical bone slices were obtained from Boineslices.com (Jelling, Denmark). Cell Counting Kit-8 was obtained from Dojindo Molecular Technologies (Dojindo, Japan). The TRAP stain kit was obtained from Sigma-Aldrich (St. Louis, MO, USA). The Actin Cytoskeleton and Focal Adhesion Staining Kit was purchased from Millipore (Darmstadt, Germany). The DCFDA cellular ROS detection assay kit was obtained from ABcam (Cambridge, UK). Alpha minimal essential Medium (α-MEM) and fetal bovine serum (FBS) were purchased from Gibco (Life Technologies, Carlsbad, CA, USA). Penicillin–streptomycin solution was obtained from Hyclone (Thermo Scientific, Waltham, MA, USA). DHA was purchased from Sigma-Aldrich.

For the isolation of bone marrow-derived macrophages (BMMs), bone marrow cells (BMCs) were lysed with erythrocyte lysate buffer (Leagene, Beijing, China) and centrifuged at 1200 rpm/min for 5 min. After removal of the supernatant, BMCs were cultured overnight in complete medium (α-MEM + 10% FBS), and then the unadhered cells were collected and cultured for 2 days in complete medium supplemented with 30 ng/mL macrophage colony stimulating factor (M-CSF) (PeproTech, Hamburg, Germany). BMMs were then inoculated into 96-well culture plates (8000 cells/well) and cultured in complete medium containing 30 ng/mL M-CSF and 50 ng/mL receptor activator ligand of NF-kB (RANKL) (PeproTech) for 4 days (the medium was changed every 2 days). Tartrate-resistant acid phosphatase (TRAP) activity was detected with a TRAP Stain Kit (Sigma-Aldrich, St. Louis, MI, USA) according to the manufacturer’s protocol, and the mRNA of osteoclastic markers were detected by real-time quantitative reverse transcription-PCR (qRT-PCR).

Epothilone B (Epo B) was purchased from MedChemExpress (Princeton, NJ). Recombinant Mouse M-CSF and Recombinant Mouse RANKL were obtained from R&D Systems (Minneapolis, MN). TRAP stain kit and Lipopolysaccharide from Escherichia coli O55:B5 were purchased from Sigma-Aldrich (St. Louis, USA). Antibodies against c-FOS, NFATc1, IL-6 and gp130 were from Cell Signaling Technology Inc (Danvers, USA). Antibodies against phospho-NFκB p65, NF-κB p65, phospho-IκBα, IκBα, STAT3, phospho-STAT3 (Tyr705) and β-actin were from Bioworld Technology (St. Louis Park, MN, USA). Antibody against Akt, phospho-Akt (Ser473), PI3K, phospho-PI3K (Ser1070), MMP9, CD9 and CTSK were purchased from bioss (Beijing, China). Nitric oxide detection kit (Beyotime Biotechnology, Shanghai, China). CCK-8 kit was purchased from Dojindo Molecular Technologies (Tokyo, Japan). The Focal Adhesion and Actin Cytoskeleton Staining Kit was obtained from Millipore (Darmstadt, Germany). Bovine cortical bone slices for pit formation assay were obtained from Boineslices.com (Jelling, Denmark). Dulbecco’s modified eagle medium (DMEM), alpha minimal essential medium (α-MEM), and fetal bovine serum (FBS) were purchased from Gibco (Life Technologies, USA). RAW264.7 cells (mouse macrophage cells) were obtained from the American Type Culture Collection (Rockville, MD, USA).

Antibodies against Histone 3 (bs-17422R), C1QC (bs-11336R), H2B (bs-52099R), C3B (bs-4871R), GAPDH (bs-2188R), β-tubulin (bs-0715R) were all purchased from Bioss Antibodies (Beijing, China). Antibody against RANK (#119805) was obtained from BioLegend (California, USA), while antibody against Annexin-V(#AMM01981G) was obtained from BD Biosciences (New Jersey, USA). Fetal bovine serum (FBS) was purchased from CELLCOOK (Guangzhou, China) and penicillin-streptomycin were purchased from Gibco (Thermo Scientific, MA, USA). Recombinant mouse RANKL was purchased from R&D Systems (Minneapolis, MN, USA). TRAP stain kit was obtained from Sigma-Aldrich (NY, USA). Annexin V and PI Apoptosis Kit (F6012) was purchased from US Everbright® Inc. (Suzhou, China). Staurosporine was purchased from Med Chem Express (New Jersey, USA).