β catenin

The reagents used for cell treatment were Lipofectamine 3000 (L3000015, Invitrogen), ascorbic acid (A4544, Sigma-Aldrich), sodium β-glycerophosphate (G9422, Sigma-Aldrich), and dexamethasone (D4902, Sigma-Aldrich). Primary antibodies included PER2 (NBP2-24616, Novus Biologicals), BMAL1 (NB100-2288, Novus Biologicals), PPARγ (A0270, ABclonal Technology, China), AKT1-Ser473 (AP0140, ABclonal Technology, China), β-catenin-Ser552 (AP0579, ABclonal Technology, China), AKT1 (A11016, ABclonal Technology, China), AMELX (ab153915, Abcam), β-catenin (A11343, ABclonal Technology, China), LAMIN B1 (A1910, ABclonal Technology, China), β-tubulin (PMK081M, BioPM, China), GAPDH (PMK042M, BioPM, China), CK14 (MA5-11599, ThermoFisher Scientific), F-actin (ab205, Abcam), and E-cadherin (sc-8426, Santa Cruz).

Proteins were extracted from the lysed cells with a Mem-PER Plus membrane protein extraction kit (Thermo Fisher Scientific, catalog no. 89842). Trans-Blot Turbo (Bio-Rad) was used to transfer proteins from 8% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gel to a polyvinylidene difluoride Western blot membrane. The transferred membrane was incubated with 3% BSA and then with the primary antibodies at 4 °C overnight, including phospho-β-catenin-Ser⁶⁷⁵ (ABclonal, catalog no. AP0795), phospho-myosin light chain 2 (Ser¹⁹) (Cell Signaling, catalog no.3671), β-catenin (ABclonal, catalog no. A0316), tubulin (Abcam, catalog no. ab7291), APC (Abcam, catalog no. ab40778), Oct4 (Abcam, catalog no. ab181557), and Bmi1 (Abcam, catalog no. ab126783). The membrane was washed and incubated with the secondary antibodies, goat anti-mouse IgG (H+L)-HRP (horseradish peroxidase) conjugate and goat anti-rabbit IgG (H+L)-HRP conjugate (Bio-Rad, catalog no. 1706516), for 2 h. GAPDH (Abcam) was used for reference. The membranes were then incubated with Clarity and Clarity Max Western ECL Blotting Substrates (Thermo Fisher Scientific). Images were captured using the ChemiDoc Imaging system (Thermo Fisher Scientific).

HFLS-RA cells were cultured overnight in a 6-well plate with complete medium. Then, the cells were incubated with XF (3, 4, 5 mg/mL) for 24 hours. Finally, RIPA buffer containing protease inhibitor phenylmethanesulfonyl fluoride (Beyotime, Shanghai, China) was used to lyse the cells, and centrifuged and the supernatant was collected. The protein concentration of the supernatant was detected by a BCA Protein Assay Kit (Beyotime, Shanghai, China). The appropriate protein concentration was adjusted with the lysate, and mixed with the loading buffer, equal concentrations of proteins were separated by SDS-PAGE and electro-transferred to a nitrocellulose membrane (NC, Beyotime, Shanghai, China, FFN08). The latter NC membranes were blocked with 5% nonfat milk for 1 hour with shaking. Then, the membranes were incubated with primary antibodies including NF-κB p65 (ABclonal, Wuhan, China, A10609), β-catenin (ABclonal, Wuhan, China, A11512) and GAPDH (ABclonal, Wuhan, China, AC001) overnight at 4 °C. The NC membranes were rinsed with TBST thrice and incubated with secondary antibodies for 1h at room temperature. The expression of GAPDH was used for normalizing the proteins expressions of different groups. Bands were detected using a 2-color infrared laser imaging system (Odyssey Clx Image Studio 3.1). The experiments were repeated 3 times.

Mouse tumour tissues were fixed with 4% paraformaldehyde for 24 h and embedded in paraffin. Sections (5 μm-thick) were prepared and blocked with Immunol Staining Blocking Buffer (P0102; Beyotime) and 5% goat serum (C0265; Beyotime) followed by incubation with primary antibodies against VIM (cat. no. 5741; Cell Signaling Technology), E-cadherin (cat. no. A3044; ABclonal), β-catenin (cat. no. 8480; Cell Signaling Technology) and c-Myc (cat. no. ab32072; Abcam) at 4 °C overnight. These sections were then washed and incubated with the mouse/rabbit streptomycin-biotin assay system (SP-9000; ZSGB-BIO, Beijing, China). Expression of vimentin and E-cadherin were visualised by 3,3′-diaminobenzidine tetrahydrochloride (ZLI-9017; ZSGB-BIO) staining. At least four random fields were examined for each sample. Images were captured using a microscope (IX73, Olympus).

Cellular protein of osteoblasts from rats was extracted with RIPA lysis buffer (#P0013B, Beyotime, China), and BCA kit (#P0012S, Beyotime, China) was adopted to determine the protein concentration. Subsequently, the SDS-PAGE gels were prepared according to the SDS-PAGE kit (#PG112, Epizyme, China). The electrophoresis condition was set at 80 V for 1 h and then was adjusted to 120 V for 40 min. The membrane transfer condition was settled at 250 mA for 2 h. Then, 5% skimmed milk was used for blocking at 25°C for 1 h. Primary antibodies PTGS2 (#A1253, ABclonal, China), β-catenin (#A19657, ABclonal, China), TCF4 (#A1141, ABclonal, China), ALP (#A19286, ABclonal, China), and GAPDH (#A19056, ABclonal, China) (dilutions of 1 : 1000, 1 : 500, 1 : 500, 1 : 500, and 1 : 100, respectively) were added for incubation overnight at 4°C. The membranes were washed 3 times with TBST, 10 min each time. Next, secondary antibody (#AS014, ABclonal, China) was added to the membranes and incubated at room temperature for 1 h. Then, the membranes were rinsed with TBST 3 times, 10 min each time, and exposed using ECL exposure liquid. The gray values of the target bands were analyzed and calculated by ImageJ (version 1.8.0, USA).