Anti dmp1

Tissue sections or cells were fixed, washed and blocked, and then incubated with the following primary antibodies: anti-Nestin (1/200; Abcam), anti-NF200 (1/200; Abcam), anti-MBP101 (1/200; Abcam), anti-Active β-catenin (1/200; Cell Signaling Technology), anti-DSPP (1/200; Zen Bioscience), anti-DMP1 (1/200; Santa Cruz Biotechnology), anti-CD31 (1/200; Zen Bioscience), and anti-VEGF (1/200; Abcam). The goat anti-rabbit 488 (1/200; Invitrogen) and goat anti mouse 488 (1/200; Invitrogen) secondary antibodies were used. Sections or cells were counterstained with DAPI, mounted with fluorescent mounting media and visualized using a fluorescence confocal microscope (Olympus FV1200, Olympus). Accordingly, quantitative analysis of the staining was conducted by measuring the average optical density using the ImageJ software. At least 3 replicates were performed.

Proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). Membranes were blocked with 4% skim milk in Tris-buffered saline containing 0.1% Tween 20 at room temperature. Proteins were detected with primary antibodies anti-DMP1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-RUNX2 (MBL, Woburn, MA, USA), anti-BMP2 (Abcam, Cambridge, UK), anti-OCN (Abcam), and anti-β-actin (Santa Cruz Biotechnology), followed by incubation with horseradish peroxidase-conjugated secondary antibodies (Enzo Life Sciences, Minneapolis, MN, USA). It was detected using an enhanced chemiluminescence detection system (GE Healthcare, Buckinghamshire, UK), according to the manufacturer's instructions. Signals were detected using a Fusion Solo X (Vilber, Paris, France). The protein expression levels were normalized to that of β-actin. All Western blot analyses were repeated three times under the same conditions.

Glass coverslips were sterilized by dipping them in 90% ethanol, and then carefully drying them over a flame. Then, a coverslip was placed in each well of a sterile 6-well tissue culture plate. Cell suspensions containing 1 × 10⁴ cells/mL were added to each coverslip. After incubating the cells for 24 h, the medium was switched to material extract. After exposure to the extract medium for 7 days, cells were fixed in 4% paraformaldehyde for 20 min at room temperature. Then, they were incubated in 0.1% Triton X-100 in PBS for 15 min. After blocking with 10% goat serum for 1 h at room temperature, cells were incubated for 2 h with monoclonal mouse anti-DSPP (Santa Cruz Biotechnology), anti-DMP1 (Santa Cruz Biotechnology), or anti-ON (Santa Cruz Biotechnology) (1:100) in 10% goat serum. Then, the cells were incubated with fluorophore-conjugated secondary antibodies (anti-mouse-FITC) for 2 h at room temperature. Coverslips were mounted onto slides using mounting solution. Fluorescent images were obtained using a fluorescence microscope (Carl Zeiss, Jena, Germany).

For histology and immunohistochemistry analysis, sections of 6 µm were made for hematoxylin/eosin staining. Immunohistochemical staining was performed as previously described 35 (link). Primary anti-DSPP (1/200; Zen Bioscience), anti-DMP1 (1/200; Santa Cruz Biotechnology), and anti-OCN (1/200; Zen Bioscience) antibodies were used. Detection of crossreacted secondary antibodies was performed using the DAB kit (Gene Tech, China). Accordingly, quantitative analysis of the staining was conducted by measuring the average optical density using the ImageJ software. At least 3 serial sections from each sample were examined, and at least 3 samples were included in each group.

The automated western blot was performed using Simple Wes (Protein Simple, USA) following the manufacturer’s protocol. Briefly, 1.5 μg of protein from the cell lysates or exosomes was added to the standard fluorescent mastermix, then was loaded into corresponding wells of the prefilled Wes assay plate, along with antibody diluent (Protein Simple, USA), anti-DSP (Santa Cruz, USA), anti-DMP1, anti-RUNX2, anti-ALP, anti-CD9, anti-CD63, anti-TGFβ1, anti-TGFR1, anti-Smad2/3, anti-p-Smad2/3, anti-Smad4, anti-β-Tublin (Affinity, USA), anti-LTBP1 (Affinity, USA), anti-rabbit secondary antibody (Protein Simple, USA), and Streptavidin-HRP, followed by luminal peroxide mix. The imaging and analysis were done with compass software (Protein Simple, USA).