Journal: British Journal of Cancer

Article Title: Breast osteoblast-like cells: a new biomarker for the management of breast cancer

doi: 10.1038/s41416-018-0255-y

Figure Lengend Snippet: Breast osteoblast-like cells (BOLCs) and the expression of breast cancer biomarkers. a Graph shows the Nottingham histological score of breast-infiltrating carcinomas. b Graph displays the number of BOLCs in G1, G2 and G3 groups (Nottingham score). c Image shows BOLCs in infiltrating breast cancer; RANKL expression Texas-Red, RUNX2 expression FITC (scale bar represents 20 µm). d High magnification of BOLCs; RANKL expression Texas-Red, RUNX2 expression FITC (scale bar represents 50 µm). e Graph displays the correlation between BOLCs and the number of TGFβ-positive cells ( p < 0.0001; R 2 0.865). Representative image of TGFβ cancer-positive cells in a breast-infiltrating carcinoma (scale bar represents 100 µm). f Graph shows the correlation between BOLCs and the number of vimentin-positive cells ( p < 0.0001; R 2 0.810). Representative image of vimentin cancer-positive cells in a breast-infiltrating carcinoma (scale bar represents 80 µm). g Graph displays the correlation between BOLCs and the percentage of Ki67-positive cells ( p < 0.0001, R 2 0.859). Representative image of Ki67 cancer-positive cells in a breast-infiltrating carcinoma (scale bar represents 100 µm). h Graph shows the correlation between BOLCs and the number of CD44-positive cells ( p < 0.0001, R 2 0.627). Representative image of CD44 cancer-positive cells in a breast-infiltrating carcinoma (scale bar represents 100 µm). i Graph displays the correlation between BOLCs and the number of ER-positive cells ( p < 0.0001, R 2 0.827). Representative image of ER cancer-positive cells in a breast-infiltrating carcinoma (scale bar represents 100 µm). j Graph shows the number of BOLCs in score 0, score 1, score 2 and score 3 groups (Her2 scoring system) ( p = 0.581). Representative image of HER2 cancer-positive cells in a breast-infiltrating carcinoma (scale bar represents 100 µm)

Article Snippet: Sections were then incubated for 1 h at room temperature with the following primary antibodies diluted 1:100: RUNX2 (clone 1D8, Novus Biologicals, USA), RANKL (clone 12A668, Abcam, Cambridge, UK) vimentin (clone 2D1, Novus Biologicals), TGFβ (clone 1D11.16.8, Ki67, Novus Biologicals), CD44 (clone 8E2F3, Novus Biologicals), ER (clone SP1, Novus Biologicals), and HER2 (clone 4B5, Ventana, Tucson, USA).

Techniques: Expressing

Journal: Dentistry journal

Article Title: Evaluation of Two Configurations of Hydroxyapatite and Beta-Tricalcium Phosphate in Sinus Grafts with Simultaneous Implant Installation: An Experimental Study in Rabbits.

doi: 10.3390/dj11050121

Figure Lengend Snippet: Figure 9. Photomicrographs of the maxillary sinus region of rabbits 40 days after filling with HA + β-TCP in the “paste and granule” configurations and concomitant implant installation, showing immunolabeling of RUNX2, VEGF, OCN, and TRAP proteins. The black arrows indicate examples of immunolabeling.

Article Snippet: The primary antibodies used were polyclonal antibodies, produced in goats, against RUNX2 (SC8566), VEGF (SC1881), OCN (SC18319) and TRAP (SC 30832) (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Immunolabeling

Journal: FEBS Open Bio

Article Title: In vitro evaluation of periapical lesion‐derived stem cells for dental pulp tissue engineering

doi: 10.1002/2211-5463.13336

Figure Lengend Snippet: Primers used in the qRT‐PCR.

Article Snippet: The membranes were blocked with 5% non‐fat milk and incubated with primary antibodies against RUNX family transcription factor 2 (RUNX2) (dilution 1 : 500; #12556S; Cell Signaling Technology, Danvers, MA, USA), dentin sialophosphoprotein (DSPP) (dilution 1 : 500; sc‐73632, Santa Cruz Biotechnology, Santa Cruz, CA, USA), dentin matrix protein 1 (DMP1) (dilution 1 : 500; ab103203; Abcam, Cambridge, UK), Sp7 transcription factor (SP7) (dilution 1 : 1000; PA5‐115697; Thermo Fisher), VEGF (dilution 1 : 100; MA5‐13182; Thermo Fisher), GDNF (dilution 1 : 200; 711074; Thermo Fisher) and GAPDH (WB0197; Biotechwell) and were then incubated with HRP‐conjugated secondary antibodies (dilution 1 : 5000; Biotechwell).

Techniques: Sequencing

Journal: FEBS Open Bio

Article Title: In vitro evaluation of periapical lesion‐derived stem cells for dental pulp tissue engineering

doi: 10.1002/2211-5463.13336

Figure Lengend Snippet: Comparison of the osteo/odontogenic capacities of the cells. (A) ALP staining after osteo/odontogenic induction for 3 and 7 days. Scale bar = 500 μm. (B) Alizarin red staining showing the presence of calcified nodules after 7, 14, 21 and 28 days of osteo/odontogenic induction. Scale bar = 500 μm. (C) Gene expression patterns of ALP , RUNX2 and DSPP after osteogenic induction for 3 and 7 days; the expression level was normalized to that of ACTB ( n = 3, Student’s t ‐test). * P < 0.05, ** P < 0.01. (D) Western blotting detection (upper) and imagej analysis (below) of DSPP, DMP1, RUNX2 and SP7 after osteogenic induction for 7, 14 and 21 days. Data are shown as the mean ± SD.

Article Snippet: The membranes were blocked with 5% non‐fat milk and incubated with primary antibodies against RUNX family transcription factor 2 (RUNX2) (dilution 1 : 500; #12556S; Cell Signaling Technology, Danvers, MA, USA), dentin sialophosphoprotein (DSPP) (dilution 1 : 500; sc‐73632, Santa Cruz Biotechnology, Santa Cruz, CA, USA), dentin matrix protein 1 (DMP1) (dilution 1 : 500; ab103203; Abcam, Cambridge, UK), Sp7 transcription factor (SP7) (dilution 1 : 1000; PA5‐115697; Thermo Fisher), VEGF (dilution 1 : 100; MA5‐13182; Thermo Fisher), GDNF (dilution 1 : 200; 711074; Thermo Fisher) and GAPDH (WB0197; Biotechwell) and were then incubated with HRP‐conjugated secondary antibodies (dilution 1 : 5000; Biotechwell).

Techniques: Comparison, Staining, Gene Expression, Expressing, Western Blot

Journal: IEEE Open Journal of Nanotechnology

Article Title: Osteogenic Effect of Rabbit Periosteum-Derived Precursor Cells Co-Induced by Electric Stimulation and Adipose-Derived Stem Cells in a 3D Co-Culture System

doi: 10.1109/ojnano.2021.3131653

Figure Lengend Snippet: FIGURE 7. Double immunofluorescence staining showing protein expressions of the PDPCs after receiving ES treatment and co-culturing with ADSCs for 2 days. The ES treatment conditions were 0.7 v/cm, 80 kHz, and 3 h/day. The cell nuclei was stained by DPAI fluorescence (blue). The RUNX2 protein was stained by FITC fluorescence (green). The OPN protein was stained by iFlour 594 fluorescence (red).

Article Snippet: After washing, the cell-hydrogel construct was incubated with primary antibodies including diluted FITC-conjugated rabbit anti-RUNX2 polyclonal antibody (ARP36679; AVIVA SYSTEMS BIOSYSTEM, USA) and mouse anti-OPN monoclonal antibody (MBS555003; MyBioSource, USA) in PBS for 2 h at room temperature or overnight at 4 °C avoiding from the light.

Techniques: Staining

Journal: Molecular Medicine Reports

Article Title: LINC01638 silencing inhibits cancer cell proliferation in colorectal adenocarcinoma through interaction with RUNX2

doi: 10.3892/mmr.2019.10191

Figure Lengend Snippet: Plasma RUNX2 mRNA levels are upregulated in colorectal adenocarcinoma patients and are positively correlated with the levels of lncRNA LINC01638. (A) Plasma RUNX2 mRNA levels were upregulated in colorectal adenocarcinoma patients compared to healthy controls (*P<0.05). Plasma levels of RUNX2 mRNA and lncRNA LINC01638 were positively correlated in (B) colorectal adenocarcinoma patients but not in (C) healthy controls.

Article Snippet: Antibodies used in this study included primary antibodies of rabbit anti-human RUNX2 (1:1,500; cat. no. ab23981; Abcam, Shanghai, China) and (1:1,500; cat. no. ab9485; Abcam) and secondary antibody goat anti-rabbit IgG-HRP (1:1,200; cat. no. MBS435036; MyBioSource, San Diego, CA, USA).

Techniques:

Journal: Molecular Medicine Reports

Article Title: LINC01638 silencing inhibits cancer cell proliferation in colorectal adenocarcinoma through interaction with RUNX2

doi: 10.3892/mmr.2019.10191

Figure Lengend Snippet: LINC01638 shRNA silencing mediates downreguatin of RUNX2 in cells of WiDr and HT-29 human colorectal adenocarcinoma cell lines. (A) LINC01638 shRNA silencing led to significantly downregulated expression of RUNX2 protein in cells of WiDr and HT-29 human colorectal adenocarcinoma cell lines, while (B) RUNX2 overexpression failed to significanly affect the expression of LINC01638 (*P<0.05).

Article Snippet: Antibodies used in this study included primary antibodies of rabbit anti-human RUNX2 (1:1,500; cat. no. ab23981; Abcam, Shanghai, China) and (1:1,500; cat. no. ab9485; Abcam) and secondary antibody goat anti-rabbit IgG-HRP (1:1,200; cat. no. MBS435036; MyBioSource, San Diego, CA, USA).

Techniques: shRNA, Expressing, Over Expression

Journal: Molecular Medicine Reports

Article Title: LINC01638 silencing inhibits cancer cell proliferation in colorectal adenocarcinoma through interaction with RUNX2

doi: 10.3892/mmr.2019.10191

Figure Lengend Snippet: LINC01638 shRNA inhibits cancer cell proliferation possibly through RUNX2. LINC01638 shRNA silencing (shRNA) significantly inhibited, while RUNX2 overexpression (RUNX2) significantly promoted the proliferation of cells of both (A) WiDr and (B) HT-29 human colorectal adenocarcinoma cell lines. In addition, RUNX2 overexpression partially reversed the advese effect of LINC01638 shRNA silencing on cancer cell proliferation (*P<0.05).

Article Snippet: Antibodies used in this study included primary antibodies of rabbit anti-human RUNX2 (1:1,500; cat. no. ab23981; Abcam, Shanghai, China) and (1:1,500; cat. no. ab9485; Abcam) and secondary antibody goat anti-rabbit IgG-HRP (1:1,200; cat. no. MBS435036; MyBioSource, San Diego, CA, USA).

Techniques: shRNA, Over Expression

Journal: Bioengineering

Article Title: An Injectable, Osteoconductive Gelatin-Enabled GelMA/HAp Hydrogel Scaffold for Minimally Invasive Bone Tissue Engineering

doi: 10.3390/bioengineering13020139

Figure Lengend Snippet: ( a ) Immunofluorescence images of Runx2 in BMSCs co-cultured with hydrogels during osteogenic differentiation. ( b ) Number of Runx2-positive cells in BMSCs co-cultured with hydrogels. *** p < 0.005; **** p < 0.0001.

Article Snippet: The fixed samples were permeabilized and blocked, followed by incubation with a primary antibody against Runx2 (Shanghai, China, Proteintech, 20700-1-AP) and then with the corresponding fluorophore-conjugated secondary antibody (USA, Thermo Fisher Scientific, A-11011).

Techniques: Immunofluorescence, Cell Culture

Journal: Bioengineering

Article Title: An Injectable, Osteoconductive Gelatin-Enabled GelMA/HAp Hydrogel Scaffold for Minimally Invasive Bone Tissue Engineering

doi: 10.3390/bioengineering13020139

Figure Lengend Snippet: ( a – d ) qPCR analysis of OCN , COL-1 , Runx2 and Osterix in BMSCs on Day 7 and 14. * p < 0.05; **** p < 0.0001.

Article Snippet: The fixed samples were permeabilized and blocked, followed by incubation with a primary antibody against Runx2 (Shanghai, China, Proteintech, 20700-1-AP) and then with the corresponding fluorophore-conjugated secondary antibody (USA, Thermo Fisher Scientific, A-11011).

Techniques:

Journal: Journal of Orthopaedic Surgery and Research

Article Title: Comparative analysis of titanium coating on cobalt-chrome alloy in vitro and in vivo direct metal fabrication vs. plasma spraying

doi: 10.1186/s13018-020-02108-4

Figure Lengend Snippet: Immunofluorescent staining of Runx-2 and fibronectin expression in osteoblasts. Stained with reds are Runx-2 and fibronectin. TPS-coated surface stained with fibronectin is showed in a and b while fibronectin staining of DMF surface is showed in c and d . Runx-2 expression of TPS surfaces is showed in e and f while that of DMF surface are showed in g and h . Blue stains are of DAPI, which were used as counterstain. Overall expression within the set area is shown in a , c , e , and g . The thickness of the stained layer is shown in b , d , f , and h

Article Snippet: After 21 days of incubation, irrigation with PBS three times, and stabilization with 4% paraformaldehyde for 10 min, the cells were incubated to use primary antibodies to RUNX-2 and fibronectin (1:100, Abcam, Cambridge, England) overnight at 4 °C.

Techniques: Staining, Expressing

Journal: Bone Research

Article Title: BMP-induced Atoh8 attenuates osteoclastogenesis by suppressing Runx2 transcriptional activity and reducing the Rankl/Opg expression ratio in osteoblasts

doi: 10.1038/s41413-020-00106-0

Figure Lengend Snippet: Atoh8 loss in osteoblasts accelerates Runx2 expression, osteoblastic differentiation, and the Rankl/Opg expression ratio. a – c Mouse primary osteoblasts were stimulated with or without 100 ng·mL −1 BMP-6 and subjected to ALP staining on day 4, von Kossa staining on day 11, and RT-qPCR on day 6 for the indicated genes ( n = 3). Values were normalized to Hprt1 . ST-2 ( d , e ) or MC3T3-E1 ( f , g ) cells were transfected with the indicated siRNA, stimulated with or without the indicated concentrations of BMP-6 for 2 days, and subjected to RT-qPCR for the indicated genes. Values were normalized to Hprt1 ( n = 3). h , i ST-2 cells were transfected with the indicated expression plasmid, stimulated with or without 100 ng·mL −1 BMP-6 for 2 days, and subjected to RT-qPCR. Values were normalized to Hprt1 ( n = 3). The data shown are the mean ± SD. n.s. not significant; * P < 0.05; ** P < 0.01; *** P < 0.001

Article Snippet: The primary antibodies used in this study were mouse monoclonal anti-Runx2 (1:200, D130-3, MBL, Nagoya, Japan), rabbit polyclonal anti-Osterix (Osx) (1:500, ab22552, Abcam, Cambridge, UK), rabbit polyclonal anti-IBSP (Bsp) (1:50, LS-C190916, LSBio, Seattle, WA, USA), rabbit polyclonal anti-Osteocalcin (Ocn) (1:100, ab93876, Abcam), mouse monoclonal anti-RANKL (1:200, ab45039, Abcam), and rabbit polyclonal anti-Opg (1:100, ab183910, Abcam) antibodies.

Techniques: Expressing, Staining, Quantitative RT-PCR, Transfection, Plasmid Preparation

Journal: Bone Research

Article Title: BMP-induced Atoh8 attenuates osteoclastogenesis by suppressing Runx2 transcriptional activity and reducing the Rankl/Opg expression ratio in osteoblasts

doi: 10.1038/s41413-020-00106-0

Figure Lengend Snippet: Atoh8 silencing-mediated promotion of Runx2 expression, osteoblast differentiation, and Rankl/Opg expression is inhibited by Runx2 knockdown in ST-2 cells. a – d ST-2 cells were transfected with the indicated siRNA, stimulated with or without 100 ng·mL −1 BMP-6 for 2 days, and subjected to RT-qPCR. Values were normalized to Hprt1 ( n = 3). The data shown are the mean ± SD. n.s. not significant; * P < 0.05; ** P < 0.01; *** P < 0.001

Article Snippet: The primary antibodies used in this study were mouse monoclonal anti-Runx2 (1:200, D130-3, MBL, Nagoya, Japan), rabbit polyclonal anti-Osterix (Osx) (1:500, ab22552, Abcam, Cambridge, UK), rabbit polyclonal anti-IBSP (Bsp) (1:50, LS-C190916, LSBio, Seattle, WA, USA), rabbit polyclonal anti-Osteocalcin (Ocn) (1:100, ab93876, Abcam), mouse monoclonal anti-RANKL (1:200, ab45039, Abcam), and rabbit polyclonal anti-Opg (1:100, ab183910, Abcam) antibodies.

Techniques: Expressing, Transfection, Quantitative RT-PCR

Journal: Bone Research

Article Title: BMP-induced Atoh8 attenuates osteoclastogenesis by suppressing Runx2 transcriptional activity and reducing the Rankl/Opg expression ratio in osteoblasts

doi: 10.1038/s41413-020-00106-0

Figure Lengend Snippet: Atoh8 interacts with Runx2 and inhibits Runx2 transcriptional activity via the bHLH domain. a COS-7 cells were transfected with the indicated expression plasmids and subjected to the 6xOSE2 reporter luciferase assay ( n = 3). The protein expression level of the FLAG-tagged Atoh8 vector was monitored by immunoblotting using an anti-FLAG antibody. b COS-7 cells were transfected with the indicated expression plasmids with or without 20 μmol·L −1 MG132 for 2 h and subjected to immunoprecipitation with the anti-FLAG antibody, followed by immunoblotting analysis with the indicated antibodies. c Schematic presentation of the constructs of full-length and deletion mutants of human ATOH8 used in this study. d COS-7 cells were transfected with the indicated expression plasmids with or without 20 μmol·L −1 MG132 for 2 h and subjected to immunoprecipitation with the anti-FLAG antibody, followed by immunoblotting analysis with the indicated antibodies. e COS-7 cells were transfected with the indicated expression plasmids and subjected to a 6xOSE2 reporter luciferase assay ( n = 3). The protein expression level of the FLAG-tagged Atoh8 vectors was monitored by immunoblotting using the anti-FLAG antibody. The data shown are the mean ± SD. n.s. not significant; ** P < 0.01; *** P < 0.001

Article Snippet: The primary antibodies used in this study were mouse monoclonal anti-Runx2 (1:200, D130-3, MBL, Nagoya, Japan), rabbit polyclonal anti-Osterix (Osx) (1:500, ab22552, Abcam, Cambridge, UK), rabbit polyclonal anti-IBSP (Bsp) (1:50, LS-C190916, LSBio, Seattle, WA, USA), rabbit polyclonal anti-Osteocalcin (Ocn) (1:100, ab93876, Abcam), mouse monoclonal anti-RANKL (1:200, ab45039, Abcam), and rabbit polyclonal anti-Opg (1:100, ab183910, Abcam) antibodies.

Techniques: Activity Assay, Transfection, Expressing, Luciferase, Plasmid Preparation, Western Blot, Immunoprecipitation, Construct

Journal: Bone Research

Article Title: BMP-induced Atoh8 attenuates osteoclastogenesis by suppressing Runx2 transcriptional activity and reducing the Rankl/Opg expression ratio in osteoblasts

doi: 10.1038/s41413-020-00106-0

Figure Lengend Snippet: Illustration of this study’s results and a working model. BMP signaling enhances the Rankl/Opg expression ratio in cells of the osteoblastic lineage to promote osteoclastic bone resorption in bone remodeling. Moreover, the BMP-Smad pathway directly induces Atoh8 in immature osteoblasts. Atoh8 interacts with Runx2 and inhibits Runx2 transcriptional activity to suppress autoregulation of Runx2 and subsequent osteoblastic differentiation, as well as the Rankl/Opg expression ratio, thereby regulating the osteoclast number negatively to prevent excessive BMP-mediated bone resorption (left panel). The enhanced Runx2 activity in Atoh8-KO mice likely limited osteoblast maturation to maintain bone formation, while Runx2 increased the Rankl/Opg expression ratio to promote bone resorption and loss (right panel)

Article Snippet: The primary antibodies used in this study were mouse monoclonal anti-Runx2 (1:200, D130-3, MBL, Nagoya, Japan), rabbit polyclonal anti-Osterix (Osx) (1:500, ab22552, Abcam, Cambridge, UK), rabbit polyclonal anti-IBSP (Bsp) (1:50, LS-C190916, LSBio, Seattle, WA, USA), rabbit polyclonal anti-Osteocalcin (Ocn) (1:100, ab93876, Abcam), mouse monoclonal anti-RANKL (1:200, ab45039, Abcam), and rabbit polyclonal anti-Opg (1:100, ab183910, Abcam) antibodies.

Techniques: Expressing, Activity Assay