Alpha modified minimal essential medium α mem

Alpha‐modified minimal essential medium (α‐MEM) and foetal bovine serum (FBS) were purchased from Thermo Fisher Scientific (Scoresby). Corilagin (Figure 1A) with a purity greater than 98% was purchased from Tongtian Biotechnology Co. and dissolved in different concentrations of 0‐2 µmol/L with dimethyl sulphoxide (DMSO), respectively. Primary antibodies against ERK, JNK, p38, p65, IkBα, p‐ERK, p‐PI3K, TRAF6, p‐TAK1, p‐JNK, p‐p38, p‐p65, p‐IkBα, GAPDH, NFATc1/NFAT2, c‐Fos and c‐Src were purchased from Cell Signaling Technology, while second antibodies were purchased from Boster Biological Technology Co.. Recombinant RANKL and recombinant M‐CSF were obtained from Novoprotein Scientific Inc (Shanghai, China). Tartrate‐resistant acid phosphatase (TRAP) staining kit and all other reagents were purchased from Sigma‐Aldrich, unless stated otherwise.

NC was purchased from the National Institute for Control of Pharmaceutical and Biological Products (Beijing, China). RAW 264.7 cells were ordered from the American Type Culture Collection (Rockville, MD, USA). Murine M-CSF (mM-CSF) was obtained from R&D Systems, (Minneapolis, USA). Alpha Modified Minimal Essential Medium (α-MEM) was purchased from Thermo Fisher (Sydney, Australia) and Fetal Bovine Serum (FBS) from TRACE (Sydney, Australia). Recombinant GST-rRANKL protein was expressed and purified as previously described20 (link). Antibodies to IκBα and NFATc1 were obtained from Santa Cruz Biotechnology Com. (CA, USA). Antibody to V-ATPase d2 was generated as previously reported21 (link).

Fangchinoline with a purity ≥ 98% was purchased from Mansite (Chengdu, China). Alpha modified Minimal Essential Medium (α-MEM) and fetal bovine serum (FBS) were purchased from Thermo Fisher Scientific (Scoresby, Australia). Penicillin- Streptomycin and GlutaMAX were purchased from Thermo Fisher Scientific (Scoresby, Australia). The production and purification of recombinant RANKL were described in a previous study 9 . Antibodies to IκBα (C21), phosphorylated ERK, phosphorylated P38, NFATc1 (7A6) and β-actin were obtained from Santa Cruz Biotechnology (Paso Robles, CA, USA). Antibodies to P38, phosphorylated JNK and JNK were ordered from Cell signaling (Danvers, MA, USA). Antibody to ERK, MTS and luciferase assay kits were purchased from Promega (Madison, WI, USA). Antibody to v-ATPase-d2 was produced as reported previously 10 (link).

Magnoflorine [C₂₀H₂₄NO₄; MW, 342.41; purity, ≥98% (Meilun, Dalian, China)] was dissolved in dimethyl sulfoxide. Recombinant murine M-CSF and RANKL were from R&D Systems (Minneapolis, MN, USA). Fetal bovine serum and alpha-modified minimal essential medium (α-MEM) were supplied by Gibco-BRL (Sydney, Australia). The staining kit for tartrate-resistant acid phosphatase (TRAP) was obtained from Sigma-Aldrich (St. Louis, MO). The assay for cell viability, cell counting kit-8 (CCK-8), was from Dojindo Molecular Technology (Japan). Primary antibodies targeting extracellular signal-regulated kinase (ERK) and its phosphorylated form (phospho-ERK; Thr202/Tyr204), p38 and phospho-p38 (Thr180/Tyr182), TAK1 and phospho-TAK1, c-Jun N-terminal kinase (JNK) and phospho-JNK (Thr183/Tyr185), NF-κB (p65) and phospho-NF-κB, NF-κB inhibitor alpha (IκBα) and phospho-IκBα, c-Fos, β-actin and histone H3 (as internal controls), and appropriate secondaries conjugated to fluorescent dyes were from Cell Signaling Technology (Cambridge, MA). The primary antibody recognizing nuclear factor of activated T cells, cytoplasmic 1 (NFATc1) was supplied by Absin Bioscience Inc. (Shanghai, China). The Prime Script RT reagent kit and SYBR^® Premix Ex Taq™ II were from Takara Biotechnology (Otsu, Japan).

Jatrorrhizine hydrochloride (C₂₀H₂₀ClNO₄, Purity ≥ 98%) was purchased from Nanjing Zelang Medical Technology Co. Ltd. (Nanjing, Jiangsu, China). Alpha modified Minimal Essential Medium (α-MEM) and fetal bovine serum (FBS) were purchased from Gibco (Gibco BRL, Grand Island, NY, USA). Recombinant mouse RANKL and M-CSF were provided by PeproTech (Rocky Hill, NJ, USA). MTS reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA). TRIzol reagent was obtained from Invitrogen (Carlsbad, CA, USA). Primary anti-bodies targeting phosphorylated (p) ERK, p-p38, p-JNK1/2, ERK, p38, JNK1/2, IκBα and GAPDH were obtained from CST (Cell Signaling Technology, Inc., Beverly, MA, USA). Anti-NFATc1 and anti-c-fos antibodies were purchased from BD Biosciences (San Jose, CA, USA). Commercial kits for ALT and AST measurement were from Jiancheng Institute of Biotechnology (Nanjing, Jiangsu, China). Tartrate-resistant acid phosphatase (TRAP) staining kits were obtained from Sigma-Aldrich.
Titanium (Ti) particles (1~3 μm diameter) were purchased from Johnson Mathey Chemicals (Ward Hill, MA, USA). The particles were baked for 6 h at 180 °C, and then treated with 70% ethanol for 48 h. The sterile particles were stored in sterile PBS (300 mg∙mL⁻¹) at 4 °C until required for use.