Runx2

Cells were lysed in ice-cold RIPA buffer (0.5 M Tris-HCl, 10 mM EDTA, 1.5 M NaCl, 10% NP-40, 2.5% deoxycholic acid, pH = 7.4) supplemented with protease inhibitor cocktail (Roche, USA). Protein quantification was performed by BCA assay kit (TransGen Biotech, Beijing, China), and loaded samples containing equal amounts of protein (40 μg) were separated by 10% SDS-PAGE. Afterwards, the protein transfer onto nitrocellulose membranes (PALL, USA) was performed within transfer buffer (12 mM Tris base, 96 mM glycine, pH 8.3, and 20% methanol). Subsequently, the membranes were blocked in the TBST buffer containing 5% BSA for 2 h, followed by incubating with the antibody of SOX-2, OCT-4 (1:1000 dilution; all from Cell Signalling Technologies, CST, USA), RUNX-2, perilipin A, and SOX-9 (1:1000 dilution, all from BOSTER Biological Technology, Wuhan, China), and β-actin antibody (1:1000 dilution; TransGen, Beijing, China) overnight at 4 °C. After that, the membranes were washed with TBST buffer for three times, and incubated with secondary antibody (anti-rabbit HRP-conjugated; 1:8000 dilution; Santa Cruz Biotechnology) at room temperature for 1 h. Finally, the expression levels were analyzed by enhanced chemiluminescence and visualized by autoradiography.

Bone marrow stem cells of the two groups were cultured and adhered on small coverslips, then treated with or without 10^‐6 mol/L Dex for four days. PBS solution was used to wash the cells for 5 minutes each, then fixed with paraformaldehyde (4%) for 30 minutes, and washed again with PBS for 5 minutes. The cells were penetrated with 0.3% Triton X‐100 for 20 minutes, and washed with PBS for 5 minutes each group. Cells were kept for 30 minutes in 10% goat serum at 37°C, then were all simultaneously incubated with either both Cx43 (rabbit, 1:1000; abcam) and Runx2 (mouse, 1:300; Boster Bio) or both Cx43 and ALP (mouse, 1:300, Proteintech) for overnight at 4°C. 12 hours later, the cells of both groups were washed with PBS for three times, and then secondary antibodies (FITC‐conjugated anti‐mouse, 1:300; AlexaFluor 488‐conjugated anti‐rabbit, 1:300) were added for incubation for two hours at room temperature. Finally, DAPI was used to stain the cell nucleus for 10 minutes at room temperature. Fluorescence microscope (Zeiss, Carl Zeiss) was used to obtain the images of the cells.

RIPA buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS with complete protease inhibitor cocktail) was used for cell lysis. Total protein concentrations were evaluated using a protein assay kit (Pierce). Total protein (40 μg) was separated by electrophoresis on 8% sodium dodecyl sulfate polyacrylamide gels and then transferred to a nitrocellulose membrane. Subsequently, the membranes were blocked for 1 h at room temperature by incubation in PBS containing 5% (w/v) nonfat milk and 0.1% (v/v) Tween-20 followed by overnight incubation in the blocking buffer at 4°C with primary antibodies against ATF4 (Sigma), RUNX2 (Boster Bio, Pleasanton, CA, USA), and β-actin (Sigma). Finally, the membranes were incubated with secondary antibodies for 30 min and analyzed with an Odyssey two-color infrared laser imaging system (LI-COR Biosciences, Lincoln, NE, USA). Three separate experiments were performed. The relative density of labeled protein bands was analyzed with Image-ProPlus 5.0 software (Media Cybernetics Inc., Rockville, MD, USA).

Cells were seeded in 60 mm plastic dishes (Costar) for total protein isolation. Proteins were separated by 12% SDS-PAGE and transferred to a PVDF membrane using a semidry transfer apparatus (Hoefer) for 1.5 h at room temperature. Membranes were blocked with 5% milk in TBST for 2 h at 37°C, and incubated with primary antibodies against IRS-1 (1:1000, Cell Signaling, USA), TAZ (1:1000, Cell Signaling), RUNX2 (1:200, Boster, China), OCN (1:200, Boster) or GAPDH (1:3000, Bioworld, USA) at 4°C overnight. Membranes were incubated with IRDye800^® conjugated secondary antibody (1:20,000, Rockland, USA) for 1 h at 37°C, following scanning with the Odyssey Infrared Imaging System (Li-COR Biosciences). Then the integrated intensity for each detected band was determined with Image J, v.1.46.