Ab214821

Vascular smooth muscle cells and arterial tissues were homogenized with RIPA buffer containing proteinase and phosphatase inhibitors. The protein concentrations were quantified with Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific) according to the manufacturer's guidelines. Proteins separated by SDS‐PAGE were transferred to PVDF membranes, followed by being incubated with 5% milk overnight at 4°C. Membranes were immunoblotted using specific primary antibodies: anti‐BMP2 (1:1000, ab214821; Abcam), anti‐Runx2 (1:1000, 12556S; Cell Signaling Technology), anti‐SIRT1 (1:1000, 9475S; Cell Signaling Technology), anti‐CHOP (1:1000, 15204–1‐AP; Proteintech), anti‐ATF4 (1:1000, 10835–1‐AP; Proteintech), anti‐p21 (1:1000, ab109199; Abcam), and anti‐β‐actin (1:1000, 4970S; Cell Signaling Technology). Membranes were rinsed with PBST for three times, and then incubated with HRP‐conjugated secondary antibodies. The blots were visualized by the imaging system (Amersham Imager 600) and quantified using Image J software.

ASTX (CAS No. 472-61-7) and STZ (CAS No. 18883-66-4) were purchased from Sigma-Aldrich Co. LLC (St. Louis, MO, USA), while fetal bovine serum (FBS) was obtained from HyClone Laboratories, Inc. (Logan, UT, USA). Antibodies specific to Nrf2 (BS1258) and receptor activator of nuclear factor (NF)-κB ligand (RANKL; ALX-804-243) were purchased from Bioworld Technology, Inc. (St. Louis Park, MN, USA) and Enzo Life Sciences, Inc. (Farmingdale, NY, USA), respectively. Antibodies specific to interleukin-1β (IL-1β; sc-52012), interferon-γ (IFN-γ; sc-57208), tumor necrosis factor α (TNF-α; sc-52746), and β-actin (sc-47778) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). 2′,7′-Dichlorodihydrofluorescein-diacetate (DCF-DA), and anti-osterix (ab209484), anti-osteopontin (OPN; ab8448), anti-heme oxygenase 1 (HO-1; ab13248), anti-bone morphogenetic protein 2 (BMP2; ab214821), and anti-γ-H2AX (ab26350) antibodies were purchased from Abcam (Cambridge, UK). Unless otherwise specified, other reagents were purchased from Sigma-Aldrich Co. LLC, while laboratory consumables were from Falcon Labware (Becton-Dickinson, Franklin Lakes, NJ, USA).

The total proteins of bone tissues and osteoblasts was extracted, and the protein content was measured by BCA assay kit. The proteins were separated by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to the polyvinylidene fluoride (PVDF) membrane. After the end of the film transfer, each blot were blocked with 5% skim milk for 1 h. Then the primary antibody was added and incubated at 4°C overnight. After the incubation membrane was washed, the secondary antibody was added. ECL kit was used for photoluminescence development, and GAPDH (ab8245, abcam, United States, 1:1,000) was used as the reference protein. The grayscale values of each band were analyzed by ImageJ software. The information of primary antibodies was listed as follows: β-catenin (ab32572, abcam, United States, 1:5,000), TCF (ab185736, abcam, United States, 1:1,000), LEF-1 (ab137872, abcam, United States, 1:1,000), cyclinD (#2978, Cell Signaling Technology, United States, 1:1,500), c-myc (ab32072, abcam, Unites States, 1:1,000), Runx2 (ab236639, abcam, United States, 1:1,000), COL1A1 (ab270993, abcam, United States, 1:1,000), BMP-2 (ab214821, Abcam, United States, 1:1,000), OPN (ab228748, abcam, United States, 1:1,000).

The RIPA lysis buffer containing protease inhibitors (Beyotime Biotechnology, Shanghai, China) was utilized to extract proteins from mandible tissues and cells, and the BCA kit (San Jose, USA) was employed to examine the content of isolated proteins. After SDS-polyacrylamide gel electrophoresis, the proteins were transferred to cellulose nitrate membranes, which were then blocked with 5% skim milk for one hour and incubated with the antibodies of BMP2 (1:1000, ab214821, Abcam, MA, USA), Runx2 (1:1000, ab236639), OPN (1:1000, ab8448), OCN (1:1000, ab133612), CEBPA (1:1000, ab40761), CEBPB (1:1000, ab32358), CEBPD (1:1000, ab245414), FABP4 (1:1000, ab92501), PPARG (1:1000, ab178860), ALP (1:1000, ab229126), APN (1:1000, ab181281), Wnt (1:1000, ab219412), β-catenin (1:1000, ab32572), and β-actin (1:1000, ab8227) overnight at 4° C. After washing, the membranes were incubated with peroxidase-bound secondary antibodies for one hour at room temperature. Finally, the bands were developed with an ECL kit (Amersham Pharmacia Biotech, Little Chalfont, UK).

The expression of proteins was determined by western blot as previously described (Lin et al., 2018 (link)). Briefly, the concentration of protein was detected using a BCA kit (Beyotime, Shanghai, China). 30 μg total protein was loaded onto 8% or 12% SDS-PAGE gel and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were incubated with primary antibody at 4°C for over 12 h after blocking with 5% non-fat milk for 1 h. Subsequently, horseradish peroxidase (HRP)-conjugated goat–anti-rabbit (sc-2004, 1:5,000, Santa Cruz) or HRP-conjugated goat–anti-mouse (sc-2005, 1:5,000, Santa Cruz) secondary antibodies were used to incubate with the membrane at RT for 1 h. The immunoreactive bands were visualized using the ECL Plus Western Blot Detection Kit (Amersham Biosciences U.K. Ltd.). The relative expression levels of proteins were normalized to the intensity of the β-actin band. Primary antibodies including CD9 (ab92726, 1:1,000, Abcam), CD63 (ab68418, 1:1,000, Abcam), CD81 (ab79559, 1:1,000, Abcam), Runx2 (ab76956; 1:1,000, Abcam), Fetuin-A (16571-1-AP, 1:1,000, Proteintech), MGP (10734-1-AP, 1:1,000, Proteintech), Annexin-A2 (11256-1-AP, 1:1,000, Proteintech), BMP-2 (ab214821, 1:1,000, Abcam), Rankl (23408-1-AP, 1:500, Proteintech), and β-actin (ab6276, 1:3,000, Abcam).

To detect macrophage polarization and the expression of specific osteogenesis‐related proteins in MSCs, the immunofluorescent protein images of these cells were observed by CLSM after culturing for 3 days. Briefly, cells were fixed with 2.5% glutaraldehyde solution for 15 min, permeabilized with 0.1% Triton X‐100 for 5 min, and nonspecific binding blocked with 5% BSA for 30 min. Subsequently, macrophages in different samples were incubated with primary antibody against CCR7 (catalog no. MAB3477, R&D Systems, USA) at a 1:60 dilution at 4 °C overnight. Then, the cells were further separately incubated with Alexa Fluor 647‐conjugated CD206 antibody (dilution 1:50; catalog no. 141 712, Biolegend, USA) and Alexa Fluor 488‐conjugated antirat secondary antibody (dilution 1:500; catalog no. ab150157, Abcam, USA) for 1 h, followed by 15 min of nuclear staining with DAPI. Similarly, MSCs were incubated with primary antibody against BMP‐2 (dilution 1:100; catalog no. ab214821, Abcam, USA) and Alexa Fluor 647‐conjugated antirabbit secondary antibody (dilution 1:500; catalog no. ab150079, Abcam, USA) following the same experimental procedure. After washing with phosphate buffer saline (PBS), the nuclei and cytoskeleton were counterstained with DAPI and FITC‐labeled phalloidin, and repeatedly washed before imaging using CLSM.