rabbit polyclonal antibody directed against the human runx2  (Santa Cruz Biotechnology)

Journal: Nature cardiovascular research

Article Title: Whole-genome sequencing uncovers two loci for coronary artery calcification and identifies ARSE as a regulator of vascular calcification

doi: 10.1038/s44161-023-00375-y

Figure Lengend Snippet: a) Treatment of human coronary artery vascular smooth muscle cells (n = 6 biologically independent samples in each group) with osteogenic media decreased MMP16 mRNA expression by ~74% (left panel). Treatment of cells grown in osteogenic media with siMMP16 (resulting in >90% knockdown of MMP16 mRNA) had no effect on RUNX2 (middle panel) or CNN1 (right panel) mRNA levels. Statistical comparisons were made using a two-tailed one-way ANOVA with Sidak’s post-hoc comparison testing. The mean ± SEM is depicted in plots. b) Treatment of human coronary artery vascular smooth muscle cells grown in osteogenic media with siMMP16 had no effect on calcification, as evidenced by decreased Alizarin Red S staining.

Article Snippet: A rabbit polyclonal antibody directed against the human RUNX2 (Santa Cruz, sc-10758, 1:1000) and a rabbit monoclonal antibody directed against CNN1 (Abcam, ab46794, 1:1000) were used to detect RUNX2 and calponin protein, respectively.

Techniques: Expressing, Knockdown, Two Tailed Test, Comparison, Staining

Journal: Nature cardiovascular research

Article Title: Whole-genome sequencing uncovers two loci for coronary artery calcification and identifies ARSE as a regulator of vascular calcification

doi: 10.1038/s44161-023-00375-y

Figure Lengend Snippet: a) Cross sections of human coronary arteries from control subjects and patients with ischemic coronary artery disease (n=3 individuals in each group with 2 sections stained for each individual) were stained for ARSE (red), α-smooth muscle actin (green) and DNA (blue, DAPI). Immunofluorescence analysis shows a higher expression of ARSE in diseased arteries. Alizarin red staining for calcification was high in the coronary arteries of diseased patients with no significant stain observed in the control group. Scale bars, 200 µm for each immunofluorescence image; 500 µm for each Alizarin red staining image. Statistical comparisons were made using a two-tailed Student’s t test. The mean is depicted in plots, with the error bars representing the standard error of the mean. b) Cross sections of human coronary arteries (n=1 each for control and ischemic patient) were stained for ARSE (red), RUNX2 (green, VSMC calcification marker) and DNA (blue, DAPI). Immunofluorescence analysis shows a higher expression of ARSE in calcified diseased arteries that colocalized with increased RUNX2 expression. Scale bar 500 µm.

Article Snippet: A rabbit polyclonal antibody directed against the human RUNX2 (Santa Cruz, sc-10758, 1:1000) and a rabbit monoclonal antibody directed against CNN1 (Abcam, ab46794, 1:1000) were used to detect RUNX2 and calponin protein, respectively.

Techniques: Expressing, Control, Staining, Immunofluorescence, Two Tailed Test, Marker

Journal: Nature cardiovascular research

Article Title: Whole-genome sequencing uncovers two loci for coronary artery calcification and identifies ARSE as a regulator of vascular calcification

doi: 10.1038/s44161-023-00375-y

Figure Lengend Snippet: a) Treatment of human coronary artery vascular smooth muscle cells (n = 6 biologically independent samples in each group) with osteogenic media for 3 days increased ARSE mRNA expression approximately 5-fold. Treatment of cells grown in osteogenic media with siARSE resulted in >90% knockdown of ARSE mRNA. b) Protein expression of ARSE was measured by immunoblot (left panel) using antibodies directed against ARSE and GAPDH (for a loading control). Treatment of cells with osteogenic media increased ARSE protein levels by 1.5-fold (right panel). Treatment of cells grown in osteogenic media with siARSE resulted in >70% reduction of ARSE protein (n=4 biologically independent samples in each group). c) Treatment of cells grown in osteogenic media with siARSE ameliorated osteogenic phenotype switch as evidenced by decreased RUNX2, BGLAP, and ALPL mRNA levels, and increased CNN1 mRNA levels ~2-fold. Of note, silencing ARSE in cells grown in normal media increased CNN1 mRNA levels by > 5-fold (n=6 biologically independent samples in each group except n=5 for siARSE CNN1 data). d) Reduced ARSE expression was also associated with an approximately 50% decrease in RUNX2 and >30% increase in CNN1 protein levels assessed by immunoblot using antibodies directed against RUNX2, CNN1 and VCL (for a loading control) (n=6 biologically independent samples in each group). e) Treatment of cells grown in osteogenic media with siARSE reduced calcification by approximately 60% (right panel, n = 3 biologically independent samples in each group), as evidenced by decreased Alizarin Red staining (left panel). f) Reduced ARSE expression with siARSE treatment in human coronary artery vascular smooth muscle cells grown in collagen discs (left panel) resulted in a >3-fold increase in contraction (right panel, n=6 biologically independent samples in each group). Statistical comparisons were made using either a two-tailed one-way ANOVA with Sidak’s post-hoc comparison testing (for more than two groups) or a two-tailed Student t test (for two groups). The mean is depicted in plots, with the error bars representing the mean ± the standard error of the mean.

Article Snippet: A rabbit polyclonal antibody directed against the human RUNX2 (Santa Cruz, sc-10758, 1:1000) and a rabbit monoclonal antibody directed against CNN1 (Abcam, ab46794, 1:1000) were used to detect RUNX2 and calponin protein, respectively.

Techniques: Expressing, Knockdown, Western Blot, Control, Staining, Two Tailed Test, Comparison

Journal: Nature cardiovascular research

Article Title: Whole-genome sequencing uncovers two loci for coronary artery calcification and identifies ARSE as a regulator of vascular calcification

doi: 10.1038/s44161-023-00375-y

Figure Lengend Snippet: a) Adenoviral expression of ARSE in human coronary artery vascular smooth muscle cells was associated with an 8-fold increase in RUNX2 protein levels and an approximately 70% decrease in CNN1 protein levels, when cells were harvested 5 days after viral transduction (n=3 biologically independent samples in each group). Protein expression was determined by immunoblot (left panel) using antibodies directed against ARSE, RUNX2, CNN1 and GAPDH (for a loading control) with quantification shown in the right panel. b) As shown by Alizarin Red staining (left panel), increased ARSE expression resulted in augmented calcification in human coronary artery vascular smooth muscle cells (right panel, n = 3 biologically independent samples in each group). Two independent experiments were performed with representative images shown. c) Increased ARSE expression also caused a >70% decrease (right panel, n=6 biologically independent samples in each group) in contraction of human coronary artery vascular smooth muscle cells grown in collagen discs (left panel). Statistical comparisons were made using a two-tailed Student t test. The mean is depicted in plots, with the error bars representing the mean ± the standard error of the mean.

Article Snippet: A rabbit polyclonal antibody directed against the human RUNX2 (Santa Cruz, sc-10758, 1:1000) and a rabbit monoclonal antibody directed against CNN1 (Abcam, ab46794, 1:1000) were used to detect RUNX2 and calponin protein, respectively.

Techniques: Expressing, Transduction, Western Blot, Control, Staining, Two Tailed Test

Journal: Nature cardiovascular research

Article Title: Whole-genome sequencing uncovers two loci for coronary artery calcification and identifies ARSE as a regulator of vascular calcification

doi: 10.1038/s44161-023-00375-y

Figure Lengend Snippet: a) Treatment of human aortic vascular smooth muscle cells (n = 12 biologically independent samples in each group) with osteogenic media increased ARSE mRNA expression > 2-fold. b) Treatment of cells grown in osteogenic media with siARSE (resulting in >90% knockdown of ARSE mRNA) decreased RUNX2 (left panel), and BGLAP (middle panel) mRNA levels by ~20% and ~43% respectively, and increased CNN1 mRNA levels by > 150% (right panel). Silencing ARSE in cells grown in normal media increased CNN1 mRNA levels by > 2.5-fold. c) Treatment of cells grown in osteogenic media with siARSE reduced calcification, as evidenced by decreased Alizarin Red S staining (n=5 biologically independent samples in each group). d) Reduced ARSE expression in cells grown in collagen discs (left panel) resulted in a >3-fold increase in contraction (right panel, n=6 biologically independent samples in each group). e) Protein expression of ARSE, RUNX2 and CNN1 were confirmed by Western blot using antibodies directed against ARSE, RUNX2, CNN1 and GAPDH (for a loading control). Adenoviral expression of the 70-kDa isoform of ARSE in human aortic vascular smooth muscle cells was associated with a >15-fold increase in RUNX2 protein levels and an approximately 34% decrease in CNN1 protein levels, when cells were harvested 5 days after viral transduction (n=3 biologically independent samples in each group). f) As shown by Alizarin Red staining, increased ARSE expression resulted in augmented calcification in human aortic vascular smooth muscle cells (n=3 biologically independent samples in each group). g) Increased ARSE expression also caused a >70% decrease (right panel, n=6 biologically independent samples in each group) in contraction of human aortic vascular smooth muscle cells grown in collagen discs (left panel). Statistical comparisons were made using either a two-tailed one-way ANOVA with Sidak’s post-hoc comparison testing or a two-tailed Student t test. The mean is depicted in plots, with the error bars representing the standard error of the mean.

Article Snippet: A rabbit polyclonal antibody directed against the human RUNX2 (Santa Cruz, sc-10758, 1:1000) and a rabbit monoclonal antibody directed against CNN1 (Abcam, ab46794, 1:1000) were used to detect RUNX2 and calponin protein, respectively.

Techniques: Marker, Expressing, Knockdown, Staining, Western Blot, Control, Transduction, Two Tailed Test, Comparison

Journal: Nature cardiovascular research

Article Title: Whole-genome sequencing uncovers two loci for coronary artery calcification and identifies ARSE as a regulator of vascular calcification

doi: 10.1038/s44161-023-00375-y

Figure Lengend Snippet: Atherosclerotic vascular calcification is characterized by the phenotype switch of vascular smooth muscle cells (VSMCs) from a contractile phenotype to a proliferative, osteogenic phenotype. The osteogenic phenotype of VSMCs is characterized by decreased expression of contractile proteins such as calponin (CNN1), but increased expression of Runt-related transcription factor 2 (RUNX2), a master regulator of the phenotype switch, in addition to other markers of calcification such as bone gamma-carboxyglutamate protein (BGLAP) and alkaline phosphatase (ALPL). We identified ARSE as a major regulator of the phenotype switch.

Article Snippet: A rabbit polyclonal antibody directed against the human RUNX2 (Santa Cruz, sc-10758, 1:1000) and a rabbit monoclonal antibody directed against CNN1 (Abcam, ab46794, 1:1000) were used to detect RUNX2 and calponin protein, respectively.

Techniques: Expressing

Journal: Cells

Article Title: Mechanical Stretch Induced Osteogenesis on Human Annulus Fibrosus Cells through Upregulation of BMP-2/6 Heterodimer and Activation of P38 and SMAD1/5/8 Signaling Pathways

doi: 10.3390/cells11162600

Figure Lengend Snippet: HCS upregulates osteogenic gene expression in human AF cells. ( A , B ) AF cells were kept in static condition as controls (CL) or subjected to LCS (5%) or HCS (15%) for 8 h, and the gene expressions of Runx2, osterix, and OPN ( A ) were determined by real-time PCR analysis. The protein expressions were determined by Western blotting ( B ). ( C , D ) AF cells were subjected to HCS for the indicated times, and the gene expression of Runx2, osterix, and OPN. ( A , C ) were determined by real-time PCR analysis. ( B , D ) The protein expressions were examined by Western blotting. The results are mean ± standard deviation (SD) and are representative of three independent experiments (n = 3). Data statistics were completed with ANOVA followed by Scheffe’s test. Results were considered significant for p < 0.05.

Article Snippet: Rabbit polyclonal antibody against Runx2 was purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Gene Expression, Real-time Polymerase Chain Reaction, Western Blot, Standard Deviation

Journal: Cells

Article Title: Mechanical Stretch Induced Osteogenesis on Human Annulus Fibrosus Cells through Upregulation of BMP-2/6 Heterodimer and Activation of P38 and SMAD1/5/8 Signaling Pathways

doi: 10.3390/cells11162600

Figure Lengend Snippet: Involvement of Runx2 in tensile stretch-induced osteogenesis. AF cells were kept in static condition as CL or subjected to HCS for 8 h. Before being kept as CL or subjected to HCS, AF cells were transfected with control siRNA (si-CL), or a specific siRNA of si-Runx2. ( A ) All bar graphs represent multiple increases in mRNA expression of CL AF cells normalized to 18S rRNA. ( B ) The protein expression was examined by Western blot analyses. The results are mean ± SD and are representative of three independent experiments (n = 3). Data statistics were completed with ANOVA followed by Scheffe’s test. Results were considered significant for p < 0.05.

Article Snippet: Rabbit polyclonal antibody against Runx2 was purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Transfection, Control, Expressing, Western Blot

Journal: Cells

Article Title: Mechanical Stretch Induced Osteogenesis on Human Annulus Fibrosus Cells through Upregulation of BMP-2/6 Heterodimer and Activation of P38 and SMAD1/5/8 Signaling Pathways

doi: 10.3390/cells11162600

Figure Lengend Snippet: BMP-2/6 heterodimer mediates tensile stretch-induced osteogenic gene expression. ( A ) The AF cells were kept in static condition as the control or were subjected to HCS for 4 h, after which the BMP-2/4/6/7 mRNA expression in AF cells was determined by real-time PCR analysis. ( B , C ) AF cells were maintained as CL or subjected to 15% HCS for 8 h. Before being kept as CL or exposed to CS, AF cells were pretreated with isotype-matched IgG or neutralizing antibodies against BMP-2 (Ab-BMP-2) and BMP-6 (Ab-BMP-6). The expression levels of Runx2 and osterix mRNA ( B ) were measured by real-time PCR analysis. The protein expressions were examined by Western blotting ( C ). The results are mean ± SD and are representative of three independent experiments (n = 3). Data statistics were completed with ANOVA followed by Scheffe’s test. The results were considered significant for p < 0.05.

Article Snippet: Rabbit polyclonal antibody against Runx2 was purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Gene Expression, Control, Expressing, Real-time Polymerase Chain Reaction, Western Blot

Journal: Cells

Article Title: Mechanical Stretch Induced Osteogenesis on Human Annulus Fibrosus Cells through Upregulation of BMP-2/6 Heterodimer and Activation of P38 and SMAD1/5/8 Signaling Pathways

doi: 10.3390/cells11162600

Figure Lengend Snippet: Recombinant human (rh)BMP-2/6 heterodimer upregulates osteogenic gene expression. AF cells were kept as controls (CL) or treated with recombinant proteins of BMP-2, BMP-6, or BMP-2/6 heterodimer for 8 h, and the gene expressions of Runx2 and osterix ( A ) were measured by real-time PCR analysis. The Runx2 and osterix protein expressions were examined by Western blotting ( B ). The results are mean ± SD and are representative of three independent experiments (n = 3). Data statistics were completed with ANOVA followed by Scheffe’s test. The results were considered significant for p < 0.05.

Article Snippet: Rabbit polyclonal antibody against Runx2 was purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Recombinant, Gene Expression, Real-time Polymerase Chain Reaction, Western Blot

Journal: Cells

Article Title: Mechanical Stretch Induced Osteogenesis on Human Annulus Fibrosus Cells through Upregulation of BMP-2/6 Heterodimer and Activation of P38 and SMAD1/5/8 Signaling Pathways

doi: 10.3390/cells11162600

Figure Lengend Snippet: HCS-induced Runx2 expression is p38 MAPK dependent. AF cells were maintained in static conditions as CL or subjected to HCS for 8 h. Before being kept as CL or subjected to CS, AF cells were pretreated with DMSO, or specific kinase inhibitors for ERK1/2 (PD98059, 30 μM), JNK (SP600125, 20 μM), or p38 (SB203580, 10 μM) for 1 h and then subjected to HCS for 8 h. The mRNA ( A ) and protein ( B ) expression of Runx2 were determined by real-time PCR and Western blotting, respectively. ( C ) Control or HCS-stimulated AF cells were maintained for the times indicated, and the phosphorylation of p38 in these cells was determined using Western blotting. ( D ) The phosphorylation of p38 in AF cells after 1 h of LCS (5%) or HCS (15%) stimulation was examined using Western blotting. The results are mean ± SD and are representative of three independent experiments (n = 3). Data statistics were completed with ANOVA followed by Scheffe’s test. The results were considered significant for p < 0.05.

Article Snippet: Rabbit polyclonal antibody against Runx2 was purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Control, Phospho-proteomics

Journal: Cells

Article Title: Mechanical Stretch Induced Osteogenesis on Human Annulus Fibrosus Cells through Upregulation of BMP-2/6 Heterodimer and Activation of P38 and SMAD1/5/8 Signaling Pathways

doi: 10.3390/cells11162600

Figure Lengend Snippet: SAMD1/5/8 is involved in HCS-induced Runx2 expression. ( A ) Control or HCS-stimulated AF cells were maintained for the times indicated, and the phosphorylation of SMAD1/5/8 in these cells was determined using Western blotting. ( B ) The phosphorylation of SMAD1/5/8 in AF cells after 1 h of LCS (5%) or HCS (15%) stimulation was determined using a Western blot analysis. The results shown are representative of three independent experiments that gave similar results. ( C , D ) AF cells were kept as CL or subjected to 15% HCS for 8 h. Before being kept as CL or subjected to CS, AF cells were transfected with control siRNA (si-CL), or a specific siRNA of si-SMAD, and then exposed to HCS for 8 h. The mRNA ( C ) and protein ( D ) expression of Runx2 were examined by real-time PCR and Western blotting, respectively. The results are mean ± SD and are representative of three independent experiments (n = 3). Data statistics were completed with ANOVA followed by Scheffe’s test. The results were considered significant for p < 0.05.

Article Snippet: Rabbit polyclonal antibody against Runx2 was purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Expressing, Control, Phospho-proteomics, Western Blot, Transfection, Real-time Polymerase Chain Reaction

Journal: Cells

Article Title: Mechanical Stretch Induced Osteogenesis on Human Annulus Fibrosus Cells through Upregulation of BMP-2/6 Heterodimer and Activation of P38 and SMAD1/5/8 Signaling Pathways

doi: 10.3390/cells11162600

Figure Lengend Snippet: Inhibition of ALK3 receptor decreases HCS-induced osteogenic gene expression. AF cells were maintained in static conditions as CL or subjected to 15% HCS for 8 h. Before being kept as CL or subjected to CS, AF cells were transfected with control siRNA (si-CL), or a specific siRNA of si-SMAD, or were pretreated with DMSO, or specific kinase inhibitors for ALK3 (LDN) for 1 h, and then subjected to 15% HCS for 8 h. The gene ( A ) and protein ( B ) expression of Runx2 were examined by real-time PCR and Western blotting, respectively. The results are shown as mean ± SD. ( C ) AF cells were kept as CL or stimulated with rhBMP-2/6. Before being kept as CL or stimulated with rhBMP-2/6, AF cells were pretreated with DMSO, or specific kinase inhibitors for ALK3 (LDN) for 1 h, and then stimulated with rhBMP-2/6 for 8 h. The mRNA expression of Runx2 was measured by real-time PCR. The results are mean ± SD and are representative of three independent experiments (n = 3). Data statistics were completed with ANOVA followed by Scheffe’s test. The results were considered significant for p < 0.05.

Article Snippet: Rabbit polyclonal antibody against Runx2 was purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Inhibition, Gene Expression, Transfection, Control, Expressing, Real-time Polymerase Chain Reaction, Western Blot

Journal: Cells

Article Title: Mechanical Stretch Induced Osteogenesis on Human Annulus Fibrosus Cells through Upregulation of BMP-2/6 Heterodimer and Activation of P38 and SMAD1/5/8 Signaling Pathways

doi: 10.3390/cells11162600

Figure Lengend Snippet: Osteogenic gene expression in patients with IVD injury. The mRNA expression levels of ( A ) Runx2, ( B ) osterix, and ( C ) OPN were measured by real-time PCR. IVD and LF tissue samples were collected from patients with DDD (IVD, N:12; LF, N:12). All experiments were performed three times. Data statistics were completed with ANOVA followed by Scheffe’s test. The results were considered significant for p < 0.05.

Article Snippet: Rabbit polyclonal antibody against Runx2 was purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Gene Expression, Expressing, Real-time Polymerase Chain Reaction

Journal: International Journal of Molecular Sciences

Article Title: Effects of Obesity on Bone Healing in Rats

doi: 10.3390/ijms222413339

Figure Lengend Snippet: Influence of obesity on RUNX2 in the presence and absence of EMD. ( a ) RUNX2 protein in normal-weight control animals, HFSD-fed animals, and HFSD-fed and EMD-treated animals. Representative immunohistochemistry images are shown. ( b ) Mean intensity of RUNX2 in normal-weight control animals, HFSD-fed animals, and HFSD-fed and EMD-treated animals. Bars show mean ± SEM; n = 5 animals/group; * significant ( p < 0.05) difference between groups. ( c ) Frequency distribution of different intensity categories for RUNX2 in normal-weight control animals, HFSD-fed animals, and HFSD-fed and EMD-treated animals. The intensity of immunohistochemical staining was assigned to five intensity categories (1 = very low, 2 = low, 3 = moderate, 4 = high, 5 = very high). EMD (enamel matrix derivative), HFSD (high fat, high sucrose diet), P (pulp), D (dentin), PDL (periodontal ligament), B (bone).

Article Snippet: Sections for RUNX2 detection were pretreated with pepsin at 37 °C for 20 min, followed by pre-blocking with 1× tris-buffered saline (Merck)/4% bovine serum albumin (Merck) at room temperature for 1 h. In the next step, the sections were incubated with a rabbit polyclonal antibody against RUNX2 (ab23981, abcam, Cambridge, UK) and a rabbit polyclonal anti-osteopontin antibody (ab8448, abcam).

Techniques: Control, Immunohistochemistry, Immunohistochemical staining, Staining

Journal: Frontiers in Cardiovascular Medicine

Article Title: Organ Culture Model of Aortic Valve Calcification

doi: 10.3389/fcvm.2021.734692

Figure Lengend Snippet: Colocalisation of Alizarin Red staining with osteogenic, inflammatory and apoptotic markers. Sequential sections of a valve leaflet stimulated media alone (left column) or 100 ng/mL LPS and 3 mM phosphate (right column) for 14-days. Histochemical staining for Alizarin Red (A,B) co-localizes with regions of immunohistochemical staining for osteocalcin (C,D) , RUNX2 (E,F) , NF-kB (G,H) and caspase 3 (I,J) . Scale bar, 500 μM.

Article Snippet: Slides were then incubated overnight in a moist chamber with antibodies against rabbit polyclonal RUNX2 at 1:200 (Abcam), mouse monoclonal Osteocalcin 1:600 (Abcam), rabbit polyclonal Osteopontin 1:500 (Chemicon), mouse monoclonal NF-kB 1:800 (BD transduction), Rabbit monoclonal cleaved caspase 3 1:200 (R&D systems).

Techniques: Staining, Immunohistochemical staining

Journal: Frontiers in Cardiovascular Medicine

Article Title: Organ Culture Model of Aortic Valve Calcification

doi: 10.3389/fcvm.2021.734692

Figure Lengend Snippet: Quantification of osteogenic, inflammatory and apoptotic markers in Alizarin Red positive and negative areas of valve leaflets. Percentage area staining of 1.1 mm 2 regions from non-calcified and calcified regions for (A) osteocalcin (* P = 0.012, T -Test; n = 4), (B) RUNX2 (** P < 0.001, T -Test; n = 4), (C) NF-kB (** P < 0.001, T -Test; n = 4) and (D) caspase 3 (** P < 0.001, T -Test; n = 4).

Article Snippet: Slides were then incubated overnight in a moist chamber with antibodies against rabbit polyclonal RUNX2 at 1:200 (Abcam), mouse monoclonal Osteocalcin 1:600 (Abcam), rabbit polyclonal Osteopontin 1:500 (Chemicon), mouse monoclonal NF-kB 1:800 (BD transduction), Rabbit monoclonal cleaved caspase 3 1:200 (R&D systems).

Techniques: Staining

Journal: Frontiers in Cardiovascular Medicine

Article Title: Organ Culture Model of Aortic Valve Calcification

doi: 10.3389/fcvm.2021.734692

Figure Lengend Snippet: Effect of adenosine on the expression of osteogenic markers. Immunohistochemical staining in media alone (left column), 100 ng/mL LPS 3 μM phosphate treated (center column) and 100 ng/mL LPS 3 mM phosphate and 10 −5 M adenosine treated (right column) valve leaflets. Section were stained with RUNX2 (A–C) , osteopontin (D–F) and osteocalcin (G–I) . Scale bar, 100 μM.

Article Snippet: Slides were then incubated overnight in a moist chamber with antibodies against rabbit polyclonal RUNX2 at 1:200 (Abcam), mouse monoclonal Osteocalcin 1:600 (Abcam), rabbit polyclonal Osteopontin 1:500 (Chemicon), mouse monoclonal NF-kB 1:800 (BD transduction), Rabbit monoclonal cleaved caspase 3 1:200 (R&D systems).

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: Frontiers in Cardiovascular Medicine

Article Title: Organ Culture Model of Aortic Valve Calcification

doi: 10.3389/fcvm.2021.734692

Figure Lengend Snippet: Inhibitory effect of adenosine on calcification and expression of osteogenic markers. Effect of increasing concentrations of adenosine (10 −8 -10 −5 M) on the expression of Alizarin Red in sections of valve leaflet response to 100 ng/mL and 3 mM phosphate (* P = 0.008, T -Test; n = 8 and ** P = 0.011, T -Test; n = 6) and representative images of sections stained with Alizarin Red imaged under polarized light for each treatment group (A) . Quantification of the area of positive staining in control (media alone), 100 ng/mL and 3 mM phosphate and 100 ng/mL & 3 mM phosphate with 10 −5 M adenosine for (B) osteocalcin (** P = 0.001, * P = 0.029, ANOVA; n = 11–15 areas from 3 valves), (C) osteopontin (** P < 0.001, * P = 0.029, ANOVA; n = 13–15 areas from 3 valves) and (D) RUNX2 (* P = 0.003, ** P = 0.001, ANOVA; n = 9–14 areas from 3 valves). (E) The mean area per measurement was similar in all 3 groups.

Article Snippet: Slides were then incubated overnight in a moist chamber with antibodies against rabbit polyclonal RUNX2 at 1:200 (Abcam), mouse monoclonal Osteocalcin 1:600 (Abcam), rabbit polyclonal Osteopontin 1:500 (Chemicon), mouse monoclonal NF-kB 1:800 (BD transduction), Rabbit monoclonal cleaved caspase 3 1:200 (R&D systems).

Techniques: Expressing, Staining, Control

Journal: Regenerative Therapy

Article Title: Asperosaponin VI stimulates osteogenic differentiation of rat adipose-derived stem cells

doi: 10.1016/j.reth.2019.03.007

Figure Lengend Snippet: ASA VI promoted the expression of OCN and RUNX2. ADSCs were divided into 6 groups. Cells in Control group were incubated with DMEM/F12 containing 10% FBS. Cells in 0 M, 10 −7 M, 10 −6 M, 10 −5 M or 10 −4 M groups were cultured with osteogenic medium with 0 M, 10 −7 M, 10 −6 M, 10 −5 M or 10 −4 M ASA VI, respectively. A. ASA VI increased the levels of OCN detected by ELISA. After 3 d, 7 d, 14 d or 21 d induction, medium was collected for ELISA. B. ASA VI enhanced the mRNA levels of OCN and RUNX2 examined by qRT-PCR. After 14 d, total RNA was extracted for qRT-PCR. The relative mRNA expression of OCN and RUNX2 was normalized to GAPDH with the method of 2 −ΔΔCT . C and D, ASA VI upregulated the protein levels of OCN and RUNX2 determined by western blot. After 14 d, protein was isolated for western blot. GAPDH was employed as the internal control. *P < 0.05, compared with Control group; # P < 0.05, compared with 0 M group.

Article Snippet: After being blocked with 5% non-fat milk in Tris-buffered saline with 0.05% Tween 20 (TBST) for 1 h at room temperature, the membrane was incubated with rabbit polyclonal antibodies against OCN (1:800, catalog: A6205), RUNX2 (1:1,000, catalog: A6214) (Abclonal technology, USA), Smad2/3 (1:500, catalog: ab63672) and Smad2/3 (phospho T8) (1:1,000, catalog: ab63399) (Abcam, USA) at 4 °C overnight.

Techniques: Expressing, Control, Incubation, Cell Culture, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Western Blot, Isolation