Dmp 1

To evaluate antigen expression, immunofluorescence staining was performed after 5 days of culturing. hDPSCs were grown on round, sterilized glass cover slips (12 mm). The cells were characterized for the markers of differentiated hDPSCs (DMP-1 and β-catenin; Abcam). Briefly, the cells were washed with cold Dulbecco’s phosphate-buffered saline (PBS), fixed in 4% paraformaldehyde for 10 min, and washed twice in PBS with 0.1% Triton X-100 (PBS-T) for 5 min. Afterward, the cells were incubated with primary antibodies diluted in 1% bovine serum albumin (primary antibodies; DMP-1 and β-catenin, Abcam) overnight and then with appropriate secondary antibodies for 2 h. After washing twice with PBS-T, the nuclei were stained with DAPI (0.4 µg/mL) at room temperature for 2 min in the dark. The cells were washed twice with PBS-T, mounted on clean slide glasses with Mount Fluor (BioCyc, Potsdam, Germany), and stored at 4 °C. Representative images were captured using a Nikon Eclipse Ti microscope.

The cultured cells were extracted by RIPA (Radio Immunoprecipitation Assay) ordinary type reagent (Vazyme), which is a traditional cell tissue lysis buffer to gain proteins for western and IP experiments. Then, 30 μg of each protein was loaded to each lane, fractionated by 10% SDS-PAGE, transferred onto PVDF membranes and probed with the primary antibody of DMP1 and DSPP (Abcam, Cambridge, United Kingdom) as a target and GAPDH (CST, Boston, MA, United States) as an internal control at 4°C overnight. The secondary antibodies were added for 2 h. The blots were visualized through enhanced chemiluminescence and autoradiography.

Western blot analysis was conducted using a previously described protocol.^{39 (link)} We used antibodies against ANXA1, β-catenin, caspase 3, Lrp5, Lrp6, Runx2, Sclerostin, Snail, TGFβ, NFATc1, cathepsin K (all from Cell Signaling, Danvers, MA, USA), DMP1, ANXA6, CXCL5 (all from Abcam, Cambridge, MA, USA), M-CSF, MMP9, OPN, TPM4 (all from Santa Cruz, Dallas, TX, USA), WISP1 (R&D systems, Minneapolis, MN, USA), β-actin (Sigma, Saint Louis, MO, USA), LIMA1, Trail (both from Novus, Centennial, CO, USA), p53, CXCL1 (both from Invitrogen, Carlsbad, California, USA), and DSP (ProteinTech, Rosemont, IL, USA). The expression levels of Sclerostin and Lrp5 in CM were detected by ELISA (My BioSource, San Diego, CA, USA). Proteins isolated from A5 osteocyte CM, Y4 osteocyte CM, and osteoclast control CM (RAW264.7 cells) were analyzed with an HF Hybrid Quadrupole Orbitrap mass spectrometer. Among the 549 identified proteins, 49 proteins had higher expression levels in A5 CM than in Y4 CM and control CM. Among these proteins, 11 (p53; SPARC = osteonectin; TPM1, TPM4 = tropomyosin 1 and 4; ANXA1, ANXA6 = annexin A1 and A6; FMOD = fibromodulin; OGN = osteoglycin; DSP = desmoplakin; AHNAK = desmoyokin; and LIMA1 = LIM domain actin-binding protein 1) were identified as potential tumor suppressors.