Nfatc1

The αMEM ( alpha modification of Eagle's medium), foetal bovine serum (FBS) and DMEM were purchased from Thermo Fisher Scientific. Penicillin/streptomycin was obtained from Beyotime Institute of Biotechnology (Beyotime, Shanghai China). Schisandrin A was obtained from MCE (MedChemExpress) and was dissolved in DMSO (0.5%w/v) at the concentration of 1 mmol/L stock solution and stored at refrigerator in −20°C. The DMSO concentration was below 0.4% to the culture medium. For the animal part, Sch was diluted in corn oil before injection into C57BL/6 mice. CCK‐8 reagent was purchased from Dojindo Molecular Technologies. Primary antibodies of Cathepsin K, NFATc1, CTR, MMP‐9, TRAcP catalase and β‐actin were purchased from Proteintech Group (Rosemont, Inc). Primary antibodies of Nrf2, HO‐1, IκBα, p‐IκBα, p65, p‐p65 and c‐Fos were obtained from CST. Primary antibody of Nrf2 in animal part was obtained from Abcam. Primary antibody of CTSK and MMP9 were purchased from Proteintech Group (Rosemont, Inc). V‐ATPase‐D2 was purchased from Santa Cruz Biotechnology. Primary antibodies of RANKL and OPG were purchased from Abclonal. The dilution of antibody used in this study was 1:1000 unless noted. M‐CSF and RANKL were purchased from R&D Systems. pNFκB‐luc was obtained from Beyotime. siRNA was purchased from GenePharma. All the chemicals and reagents we used in this study were of analytical grade.

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).

Three weeks after the induction of OA, mice (n = 6 for each group) were sacrificed for western blot analysis. Protein samples were isolated from proximal tibia using a mortar and pestle. Tissues were lysed in a radioimmunoprecipitation assay (RIPA) lysis buffer, containing protease inhibitors and phosphatase inhibitors (Roche Diagnostics GmbH, Mannheim, Germany). Isolated proteins were fractionated using 10% sodium dodecyl sulfate-polyacrylamide gel and electro-transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). Primary antibodies specific to Wnt3a (Abcam, Cambridge, MA, USA), together with NFATc1, RANKL, TNF-α, Cathepsin K, p38 MAPK, NFκB p65 (Rel A) (Proteintech, Wuhan, China), Phosph-p38 MAPK, Phosph-NFκB (Cell Signaling, Danvers, MA, USA) and β-actin (Sigma, St Louis, MO, USA) were employed. After incubation with secondary IgG antibodies conjugated with horseradish peroxidase, signals were detected with enhanced chemiluminescence. Data were presented with reference to control intensities of β-actin24 .