osterix Antibody - #DF7731

Figure 3. Compound C reversed the effects of Sanguinarine on the differentiation of MC3T3-E1 cells. MC3T3-E1 cells were pretreated with 10 μM Compound C for 1 h and then exposed to 2 M Sanguinarine (SAN). Mock cells were cultured without any treatment. (A, B) At 48 h after treatment, the levels of AMPK1 and p-AMPK (A), and ALP activity in the cultured medium (B) were analyzed. (C) mRNA levels of Bmp2, Osx and Opg were detected by real-time PCR. (D) Protein levels of Bmp2, Smad1, pSmad1, Osx and Opg were determined by Western blot. ***P <0.001 versus DMSO; +++P <0.001 versus Compound C.

FIGURE 2 | The effects of MMP2-I1 on osteogenesis of hBMSCs. (H–K) The expression of RUNX2, COL1A1 and OSX proteins were determined by Western blot analysis after osteogenic differentiation for 3 and 7 days. All the data were confirmed by three repeated tests. Data were mean ± S.D.*p < 0.05 vs. the control group at the same day. #p < 0.05 vs. the control group at the same day. Scale bar = 500µm.

Fig. 3 Effects of over-expressed IGFBP3 on odontogenic and osteogenic differentiation of human dental papilla stem cells and dental mesenchyme. A IGFBP3 expression in lv5 hDPSCs versus IGFBP3-over hDPSCs after lentivirus transfection (a); Relative expression of DSPP, DMP1, OSX, OPN, OCN and ALP in lv5 hDPSCs versus IGFBP3-over hDPSCs after 0 days (b), 4 days (c) and 7 days (d) osteogenic induction. B The IGFBP3 expression response to osteogenic induction was investigated by RT-qPCR using hDPSCs. C The protein levels of IGFBP3, odontogenic and osteogenic markers in lv5 hDPSCs and IGFBP3-over hDPSCs were detected by Western blot analysis (a). Quantitative analysis of the relative protein expression in IGFBP3 (b1), DSPP (b2), OSX (b3), OPN (b4), OCN (b5) and ALP (b6). D Alizarin red staining of lv5 hDPSCs and IGFBP3-over hDPSCs with 14 days osteogenic induction; Alkaline phosphatase staining of lv5 hDPSCs and IGFBP3-over hDPSCs treated with osteogenic induction medium for 7 days. Microscopic views: a1–d1, scale bar 200 μm; gross views: a2–d2. E Microscopic views of dental mesenchymal tissue from the P1 tooth germ with lentivirus transfection (a). Relative expression of Igfbp3, Dspp, Ocn in lv5 dental mesenchyme and IGFBP3-over dental mesenchyme were detected by RT-qPCR (b). lv5, hDPSCs or dental mesenchyme with empty pGLV5 vector control; over, hDPSCs or dental mesenchyme with IGFBP3 overexpressing vector. n = 3

Figure 3 Hypoxia promotes proliferation, migration and odontoblastic differentiation in HDPCs. (A) Representative images of wound healing assays in the normoxic or hypoxic group. Scale bar, 1 mm. (B) Percentages of wound closure from three independent experiments are quantified. (C) Representative images of migratory cells stained with crystal violet. (D) Statistical quantification of the number of migratory cells from three independent experiments are shown. Scale bar, 100 µm. (E) Cell viability of HDPCs was detected using Cell Counting Kit-8 analysis. (F) ALP staining and (G) activity in the normoxic or hypoxic group on day 3. Scale bar, 200 µm. (H) Representative western blotting images showing the protein expression levels of RUNX2, Col I, OSX and OPN in the normoxic or hypoxic group on day 3, (I) which were quantified. Results are presented as the means ± SD from ≥ three independent experiments. **P<0.01 and ***P<0.001 vs. normoxia. Nor, normoxia; Hypo, hypoxia; OD, optical density; ALP, alkaline phosphatase; RUNX2, runt-related transcription factor 2; Col I, collagen type I; OSX, osterix; OPN, osteopontin.

FIGURE 2|MiR-19b-3p overexpression inhibited osteogenic differentiation of BMSCs. (A) RT-PCR was used to detect miR-19b-3p expression in BMSC-derived osteoblast after transduction. Western blot for (B) osterix andosteocalcin in BMSC-derived osteoblasts after transduction.

Figure 6 Bone formation. Protein levels of RUNX2, ostcrix, Type I collagen (A). mRNA expression level of RUNX2 (B), osterix (D), and COL1A1 (F) of the callus ares. Immunohistochemistry of RUNX2 (C), osterix (E), and type I collagen (G) within the callus area. Magnification, ×200, scale bar = 100 µm. The typical positive stained cells or regions were indicated by arrows. n=6, Mean ± SD, **p < 0.01.

Figure 6 Bone formation. Protein levels of RUNX2, ostcrix, Type I collagen (A). mRNA expression level of RUNX2 (B), osterix (D), and COL1A1 (F) of the callus ares. Immunohistochemistry of RUNX2 (C), osterix (E), and type I collagen (G) within the callus area. Magnification, ×200, scale bar = 100 µm. The typical positive stained cells or regions were indicated by arrows. n=6, Mean ± SD, **p < 0.01.

Fig. 3 A low concentration of DA facilitates PDLSCs osteogenic differentiation. (a) Flow cytometry analysis of surface markers of PDLSCs. (b) Quantitative RT-PCR analysis showing the expression of osteogenic gene expression in PDLSCs undergoing osteogenic differentiation treated with a low concentration of DA at day 7. (c) CCK-8 assay evaluating the effects of DA on PDLSC proliferation at days 3, 5, and 7. (d) Alizarin Red S staining of PDLSCs during late-stage osteogenic differentiation under various concentrations of DA (0, 10, 25, 50 nM). (e, f) Western Blot analysis of osteogenic protein expression in PDLSCs during osteogenic differentiation induced by different concentrations of DA (0, 10, 25, 50 nM). (g) Alizarin Red S staining demonstrating the osteogenic promotion by 10 nM DA in PDLSCs. Statistical significance was determined using unpaired one-way ANOVA for multiple-group comparisons.