Recombinant m csf

Berbamine (purity>99%) was purchased from MedChemExpress (New Jersey, USA). We dissolved Berbamine in dimethyl sulfoxide (DMSO) to prepare a stock solution (4 mM) and stored the stock solution at -80°C. The stock solution was further diluted with complete culture medium for in vitro experiments. For animal experiments, the stock solution was further diluted with DMSO and plant oil. A Cell Counting Kit-8 (CCK-8) was purchased from Dojindo (Tokyo, Japan). A TRAP staining kit was purchased from Sigma–Aldrich (MO, USA). Recombinant m-RANKL and recombinant M-CSF were purchased from R&D Systems (Minneapolis, MN, USA). Alpha-modified minimal essential medium (α-MEM), penicillin–streptomycin (P/S) and fetal bovine serum (FBS) were purchased from Thermo Fisher Scientific (Scoresby, Vic., Australia). Rhodamine-conjugated phalloidin and DAPI were obtained from Solarbio Co., Ltd. (Beijing, China). Universal RNA extraction kits and Evo M-MLV RT kits were purchased from Accurate Biotechnology Co., Ltd. (Hunan, China). Primary antibodies against CTSK, TRAP, MMP-9, NFATc1 (nuclear factor of activated T cells 1), CD44, DC-STAMP (dendritic cell specific transmembrane protein) and GAPDH were purchased from Proteintech (Wuhan, Hubei, China).

Asperpyrone A, with a purity >99%, was obtained from Professor RenXiang Tan in Nanjing University's (China). DMSO (dimethylsulphoxide), Trizol reagent, FBS (foetal bovine serum), a‐MEM (alpha‐modified minimal essential medium) and Ca²⁺ oscillations assay were purchased from Thermo Fisher Scientific. Recombinant M‐CSF was produced from R&D Systems, and RANKL protein (GST‐rRNAKL) was expressed and purified as previously described.12 The MTS cytotoxicity assay and luciferase report assay were purchased from Promega. The antibodies of phosphorylated ERK (p‐ERK), NFATc1, V‐ATPase‐d2, c‐fos, IκB‐α and JNK were obtained from Santa Cruz and the antibodies of P38, phosphorylated P‐38 (P‐P38), phosphorylated JNK(p‐JNK), c‐fos and ERK from Cell Signaling Technology.

BMDMs were derived from C57BL6 mice and cultured in 37°C in DMEM containing 10% FBS, 1mM sodium pyruvate, 2mM L-glutamine in the presence of 50ng/mL recombinant M-CSF (R&D Systems). AMs were derived from C57BL6 mice BALF and cultured in a complete medium.

C57BL/6J mice were acquired from Xiamen University’s Study Animal Center, and all animal investigations were authorized by Xiamen University’s Animal Care and Use Committee (XMULAC20210037). Chengdu Must Biotechnology (Chengdu, China) have supplied mogrol with a purity >98%. Dimethylsulfoxide (DMSO) was used to dissolve mogrol. Biological Industries (BI, Beit Haemek, Israel) supplied alpha-modified Eagle’s medium (α-MEM) and penicillin–streptomycin solution. Gibco (Thermo Fisher Scientific, Waltham, United States) supplemented with fetal bovine serum (FBS). Recombinant M-CSF and RANKL were acquired from R&D Systems (Minneapolis, MN, United States). Cell Signaling Technology (Danvers, United States) provided antibodies (p-ERK, ERK, p-P38, P38, p-JNK, JNK, p-P65, P65, and IκBα). Antibodies against c-FOS and TRAF6 were obtained from Abcam (Cambridge, United Kingdom). Anti-NFATc1 antibody was acquired from Santa Cruz Biotechnology (CA, United States). Anti-GAPDH antibody was purchased from Proteintech (Wuhan, China). Dojindo (Kyushu Island, Japan) provided Cell Counting Kit-8 (CCK-8). Beyotime Biotechnology, Ltd. (Shanghai, China) provided Annexin V-FITC apoptosis kits.

Bone marrow-derived macrophages (BMDM) were cultured according to described protocols 32 . Briefly, mice were euthanatized and cells from femurs were collected. 4 x 10⁵ cells were plated per sterile plastic petri dish in 10 mL of DMEM/F-12 (ThermoFisher Scientific, #11320033) supplemented with 10% fetal bovine serum (Sigma-Aldrich, #F2442), 1% penicillin/streptomycin (Sigma-Aldrich, #P4333), and 100 ng/mL of recombinant M-CSF (R&D Systems, #416-ML). Cells were incubated at 37 ºC and 5% CO₂ and the medium was changed at days 3, 5 and 7. At day 7, cells were exposed to conditioning conditions: (i) 100 ng/mL of LPS (Sigma-Aldrich, #L2880) + 20 ng/mL of rINFγ (R&D Systems, #485-MI), (ii) 20 ng/mL of rIL-4 (eBioscience), (iii) 20 ng/mL of rIL-13, (iv) 7.9 µg/mL of Oligomycin (Sigma-Aldrich, #75351) + 2.1 µg/mL of Antimycin (Sigma-Aldrich, #A8674), (v) 20 ng/mL of rIL-4 + 7.9 µg/mL of Oligomycin + 2.1 µg/mL of Antimycin, and (vi) 20 ng/mL of rIL-13 + 7.9 µg/mL of Oligomycin + 2.1 µg/mL of Antimycin. Concentrations were based on previous publications 33 (link). After 24 h, supernatants (conditioned mediums) were collected and stored at -20 ºC until use.
Motor neuron-like NSC-34 cells were cultured in DMEM (ThermoFisher Scientific, #10566016) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Sigma-Aldrich, #P-0781) in a 37 ºC and 5% CO₂ incubator.

Tetrandrine (purity >98%) was purchased from MedChemExpress (New Jersey, USA) and was dissolved in DMSO at a 10 mmol/L stock solution and stored -20℃. Further dilution was carried out in culture medium for cells and PBS medium for animals. Primary antibodies against CTSK, CTR, MMP-9, TRAF6, TRAP, GAPDH, and β-actin were purchased from Proteintech (Wuhan, Hubei, China). Primary antibodies against NFATc1, P-PI3K, AKT, P-AKT, P50, P-P50, P65, P-P65, IκBα, P-IκBα, ERK1/2, P-ERK1/2, JNK, P-JNK, P38, and P-P38 were obtained from Cell Signaling Technologies (Beverly, MA, USA). Primary antibodies against RANKL, OPG, and the ELISA kit of RANKL were obtained from ABclonal (Wuhan, Hubei, China). The ELISA kits of OPG, IL-6, TNF-α, TRAcp5B, and CTX-I were purchased from Sangon (Shanghai, China). A CCK-8 assay kit was purchased from Dojindo (Tokyo, Japan). A leukocyte acid phosphatase staining kit was obtained from Sigma‐Aldrich (MO, USA). Recombinant M‐CSF and Recombinant m-RANKL were obtained from R&D Systems (Minneapolis, MN, USA). The cell culture medium that alpha‐modified minimal essential medium (α‐MEM), fetal bovine serum (FBS), and penicillin-streptomycin were purchased from Thermo Fisher Scientific (Scoresby, Vic., Australia).