Anti osterix

Annexin V-FITC/PI detection kit was purchased from BD Biosciences (San Jose, CA, USA). Fluorometric Intracellular ROS Kit and N-acetyl-L-cysteine (NAC) were from Sigma-Aldrich (St. Louis, USA). Information of primary antibodies is listed as follows: anti-Runx2 (1 : 500, Abcam), anti-Osterix (1 : 500, Abcam), anti-OPN (1 : 1000, Abcam), anti-BSP (1 : 1000, CST), anti-OCN (1 : 500, Abcam), anti-NADPH oxidase 4 (NOX4) (1 : 500, Abcam), anti-SOD2 (1 : 500, Abcam), anti-Caspase-3 (1 : 1000, CST), and anti-GAPDH (1 : 5000, Bioworld Technology Inc., USA). ALP staining and alizarin red were both from Cyagen (Guangzhou, China). For semiquantitative analysis, p-nitrophenyl phosphate (p-NPP) and 10% cetylpyridinium chloride were from Sigma-Aldrich (St. Louis, USA). Alexa Fluor 488 conjugated was from Jackson ImmunoResearch (USA). DAPI was from Sigma-Aldrich (St. Louis, USA). For scaffold fabrication, gelatin, carboxymethyl chitosan, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), and N-hydroxysuccinimide (NHS) were all purchased from Aladdin (Shanghai, China). Calcein AM (4 mM) and Lipofectamine 3000 were from Thermo Fisher Scientific (USA).

Western blot was conducted according to a previously reported study35 (link). Total cell proteins were extracted using protein lysis buffer (Thermo Fisher Scientific Inc., Waltham, MA, USA). Total cell protein concentration was measured using a BCA kit (Pierce, Rockford, IL, USA), then equal amount of proteins were separated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), separated proteins were then transferred to polyvinylidene fluoride (PVDF) membranes (Merck KGaA, Darmstadt, Germany). The membrane was then incubated with the following primary antibodies: anti-STAT3, anti-phospho-STAT3 (Cell Signaling Technology, Inc. Danvers, MA, USA), anti-OPN, anti-Runx2, anti-Osterix, anti-SMAD4, anti-JMJD3 and anti-β-actin (All from Abcam, Cambridge, MA, USA). Then the membrane was incubated with Dylight^TM 800 secondary antibody (Invitrogen), the immunoblots were imaged by LI-COR Odyssey infrared Imaging System (LI-COR, Lincoln, NE, USA).

Whole-cell lysates were prepared on ice using 0.5 ml cold RIPA lysis buffer (Merck Millipore) containing protease inhibitor (Thermo Fisher Scientific). In brief, equal amounts of proteins (30 μg) were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to nitro cellulose membranes. After incubation with monoclonal mouse anti-PPARγ (1:250 diluted; Santa Cruz Biotechnology, CA, United States), anti-RUNX2 (1:1000 diluted; Santa Cruz Biotechnology, CA, United States), anti-COL1A1 (1:2000 diluted; Chemicon, Merck Millipore), anti-Osterix (1:1000 diluted; Abcam) and β-actin (1:5000 diluted, Chemicon, Merck Millipore) at 4°C overnight, they were further immunoblotted with HRP-conjugated horse anti-mouse IgG antibody (1:10000 diluted; Cell Signaling, United States) at 37°C for 90 min, developed with enhanced chemiluminescence (ECL) substrate (Amersham ECL Prime Western Blotting Detection Reagent, GE Healthcare) and chemiluminescence detection by ChemiDoc^TM MP Imaging System (Bio-Rad, United Kingdom). Band density was quantitated using the Image Lab^TM software Version 5.2.1 (Bio-Rad, United Kingdom).

Maxillary or mandibular bones of mice were decalcified in 10% ethylene diamine tetraacetic acid (EDTA) (pH 7.4) at 4 °C for 21 d. For immunofluorescence staining, specimens were embedded in optimal cutting temperature compound (OCT), and sectioned into 10-μm thick sections. Sections were treated with 3% hydrogen peroxide and goat serum blocking, and then they were incubated with a primary antibody. The following primary antibodies were used: anti-Osterix (1:300; Abcam, Cambridge, UK), anti-RUNX2 (1:100; Abcam), anti-type I collagen (1:200; Boster Biological Technology, Wuhan, China), anti-Amelogenin (1:100; Santa Cruz Biotechnology, Dallas, TX), anti-CD31 (1:100; Affinity Biosciences, Changzhou, China). Sections were subsequently incubated by Alexa Fluor 488 IgG (1:1000; Invitrogen) and/or Alexa Fluor 568 IgG (1:1000; Invitrogen), and counterstained with DAPI (Sigma-Aldrich).
The images were captured using a confocal microscope (Nikon, TI2-E + A1 R, Japan), and processed using the ImageJ software (US National Institutes of Health, United States).

After the tensile stress was administered for 0, 3, 6, 12, 18 and 24 h, the cells were washed twice with PBS and then harvested using TRIzol (Invitrogen) according to the manufacturer′s protocol. The protein concentration was measured using a BCA protein assay reagent kit (Beyotime, Shanghai, China). Equal aliquots of protein samples were separated using SDS-PAGE and then electrotransferred onto polyvinylidene fluoride membranes (PVDF; Millipore, Billerica, MA, USA). The membranes were then blocked with 5% lipid-free milk in Tris-buffered saline with 0.1% Tween (TBST) for 2 h at 37 °C, which was followed by an incubation with primary antibodies at 4 °C overnight. The membrane was washed with TBST 6 times for 5 min and was exposed to HRP-conjugated goat anti-rabbit secondary antibodies (Santa Cruz, Santa Cruz, CA, USA) for 2 h at 37 °C. Immunoreactive proteins were visualized using a chemiluminescence kit (Millipore). Band intensities were determined using the ChemiDoc XRS Gel documentation system and the Quantity One software (Bio-Rad, Hercules, CA, USA). Loading differences were normalized by assessing the housekeeping protein (GAPDH) in each sample. All primary antibodies, including anti-Runx2, anti-Osterix, anti-BSP, anti-OCN, anti-ALP, anti-COL-I and anti-GAPDH, were purchased from Abcam (Abcam, Cambridge, MA, USA).

Cells were seeded onto confocal dishes (20 mm in diameter) at a density of 1 × 10⁴ cells/dish. Lentivirus transfections (miR-22-NC, miR-22, miR-22-inhibitor-NC, or miR-22-inhibitor) were performed when cells reached confluence at 30–50%. Twenty-four hours after transfection, cells were exposed to 6 Gy radiation. Osteogenic induction began at 6 h after X-ray treatment and lasted for 14 days. The samples were fixed in 4% paraformaldehyde for 30 min, permeabilized with 0.5% Triton X-100 for 20 min, and blocked with 2% BSA for 30 min, respectively. Next, primary antibodies of anti-Runx2 (10 μg/ml, Abcam) and anti-Osterix (1 : 200, Abcam) were added to samples for incubation at 4°C overnight. The cells were then immersed in Alexa Fluor 488-conjugated secondary antibody for 1 h at RT. DAPI (Sigma-Aldrich, St. Louis, USA) was used to label cell nuclei for 10 min. The immunoreactive cells were visualized and captured using confocal microscopy (Leica TCS SP8, Germany). The ratio of positive cells in each sample was determined by dividing the number of immune-positive cells by the number of nuclei stained with DAPI in three random fields for each group.