Runx2

Nuclear RUNX2 expression was analysed using immunofluorescence as described previously.²⁴ The following antibodies were used: RUNX2 (1:400, #12556, Cell Signaling Technology), and secondary antibody (Alexa‐Fluor 488, 1:500, Invitrogen). DAPI (4′,6‐diamidino‐2‐phenylindole) solution was used (Sigma‐Aldrich) for nucleus stains.

Total protein was extracted from BMMs and C3H10 cells and from the bones of mice used in the OVX experiment using a radioimmunoprecipitation assay lysis buffer (RIPA) (Sigma-Aldrich), and the protein concentration was measured using a bicinchoninic acid protein (BCA) assay kit (EMD Millipore, Bedford, MA, USA). Protein equivalents were separated using 10% SDS PAGE and transferred onto PVDF membranes (EMD Millipore). The membranes were blocked for an hour at room temperature with 5% skim milk in Tris-buffered saline with Tween 20. After incubating primary antibodies overnight at 4 °C, the membranes were nurtured with corresponding HRP conjugated secondary antibodies (Proteintech Group, Chicago, IL, USA.) The bands were visualized employing ECL Luminous Liquid (EMD Millipore). ImageJ software was used to compute the relative grey level of proteins (NIH). Anti-TRAIL was procured from Santa Cruz Bio (Santa Cruz, CA, USA). Anti-CTSK, TRAP, NFATc1, OPG, RUNX2, OCN, RANKL, COL1a, p-P65, P65, p-P38, P38, p-JNK, JNK, p-ERK, ERK, p-IKBα, IKBα, IKKα, IKKβ, and p-IKKα/β were all purchased from Cell Signaling Technology (Danvers, MA, USA).

Both types of cells were lysed on ice with M-PER Mammalian Protein Extraction Reagent (Thermo Fisher Scientific, USA) supplemented with 10% protease inhibitor cocktail (Roche, Mannheim, Germany) to isolate total protein and then measured by a Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, USA) in accordance with the manufacturer’s instructions. Protein samples supplemented with loading buffer in equal proportions were electrophoresed on NuPAGE™ Bis-Tris Protein Gels (Invitrogen, Waltham, MA, USA) and then transferred onto polyvinylidene fluoride (PVDF) membranes. After incubation with 5% skim milk (5% w/v) for 2 h at room temperature, the membranes were co-incubated overnight at 4 °C with the following primary antibodies specific for GAPDH (1:1000; Cell Signaling Technology, USA), Runx2 (1:1000; Cell Signaling Technology, Danvers, MA, USA), Osx (1:1000; Abcam, Cambridge, UK), Ocn (1:2000; Abcam, UK), ELK4 (1:1000; Proteintech, Rosemont, IL, USA), GM130 (1:1000; Proteintech, USA), CD9 (1:1000; Proteintech, USA), and TSG101 (1:1000; Proteintech, USA). Next, the peroxidase-conjugated secondary antibody (1:5000; ZS-GB-BIO, Beijing, China) was utilized to treat the PVDF membranes for 2 h at room temperature. The ECL kit (Thermo Fisher Scientific, USA) was employed to visualize the signals. Densitometry analysis was conducted with ImageJ software.