Journal: Clinical Implant Dentistry and Related Research

Article Title: Bone Morphogenetic Protein ( BMP ) 9 Outperforms BMP2 in Osteogenesis and Osseointegration: In Vitro and In Vivo

doi: 10.1111/cid.70135

Figure Lengend Snippet: Comparative effects of BMP9 and BMP2 on osteogenic differentiation and osteoclastogenesis in vitro. (A) Real‐time PCR analysis of key osteogenic genes (Col1, Runx2, ALP, and OCN) in MC3T3‐E1 cells treated with 8 nM of BMP2 or BMP9 for 3, 5, and 7 days. All gene‐expression levels were normalized to GAPDH. (B) Western blot analysis of osteogenic marker proteins in cell lysates harvested after 7 days of treatment with BMP2 or BMP9. GAPDH was used as the loading control. Densitometric quantification of band intensities (integrated density) normalized to GAPDH is shown below the blots and presented as relative protein expression. (C) Western blot showing dose‐dependent p‐Smad1/5/9 in MC3T3‐E1 cells exposed to varying concentrations of BMP2 or BMP9. Phosphorylation was quantified by densitometry and expressed as fold change vs. control after normalization using [(p‐Smad1/5/9)/(total Smad1/5/9)] and further normalized to GAPDH, as shown in the graph below the blots. Asterisks indicate statistical significance for pairwise comparisons between BMP2 and BMP9 at the same concentration (****, p < 0.0001), unless otherwise indicated. (D) ALP activity and representative images of ALP staining in MC3T3‐E1 cultures after 7 days of induction with BMP2 or BMP9. (E) Alizarin Red S staining illustrating mineralized nodule formation after extended culture with BMP2 or BMP9. (F) Representative TRAP‐stained images of RAW 264.7‐derived osteoclasts following treatment with RANKL (3 nM), BMP2 (8 nM), or BMP9 (8 nM) for 5 days. TRAP‐positive multinucleated osteoclasts are indicated by arrows. Scale bar, 20 μm. (G) Quantification of TRAP‐positive multinucleated cells per well. Data are presented as the mean ± SD ( n = 3 independent experiments), and p ‐values were calculated using one‐way analysis of variance (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). BMP, bone morphogenetic protein; PCR, polymerase chain reaction; ALP, alkaline phosphatase; Col1, collagen type I; Runx2, runt‐related transcription factor 2; OCN, osteocalcin; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase.

Article Snippet: Recombinant human RANKL (11682‐HNCH; Sino Biological, Beijing, China) and a tartrate‐resistant acid phosphatase (TRAP) staining kit (MK300; Takara Bio, Shiga, Japan) were used for the osteoclast differentiation assay.

Techniques: In Vitro, Real-time Polymerase Chain Reaction, Gene Expression, Western Blot, Marker, Control, Expressing, Phospho-proteomics, Concentration Assay, Activity Assay, Staining, Derivative Assay, Polymerase Chain Reaction

Journal: PloS one

Article Title: Differential expression of the RANKL/RANK/OPG system is associated with bone metastasis in human non-small cell lung cancer.

doi: 10.1371/journal.pone.0058361

Figure Lengend Snippet: Figure 2. RANKL, RANK, and OPG mRNA expression in three NSCLC cell lines. RT-PCR was performed to detect RANKL, RANK, and OPG mRNA levels in PG-BE1, PG-LH7, and PAa cells(A). Quantitative real-time PCR revealed the relative expression of RANKL, RANK, and OPG mRNA in three NSCLC cell lines using the 22(DDCt) method(PAa cell line as a calibrator). GAPDH and b-actin were used as the internal reference(B,C). Next, the ratio of RANKL: OPG mRNA expression in three NSCLC cell lines was calculated based on Ct values for both target and reference gene(D). Bars represent the mean6the standard error of the mean (SEM) of three different experiments. **p,0.05 for PG-BE1 versus PG-LH7. *p,0.05 for PG-LH7 versus PAa. doi:10.1371/journal.pone.0058361.g002

Article Snippet: RANKL cDNA transfection Full-length human RANKL cDNA(RefSeq: NM_033012.2) was inserted into the eukaryotic vector pCMV6-XL5 (purchased from OriGene Technologies, Inc. Cat No: SC305532).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

Journal: PloS one

Article Title: Differential expression of the RANKL/RANK/OPG system is associated with bone metastasis in human non-small cell lung cancer.

doi: 10.1371/journal.pone.0058361

Figure Lengend Snippet: Figure 3. RANKL, RANK, and OPG protein expression in three NSCLC cell lines. Western blot analysis was performed to detect RANKL, RANK, and OPG protein levels in PG-BE1, PG-LH7, and PAa cells. Band intensities were normalized to b-actin (A–C). Next, the ratio of RANKL: OPG protein expression was calculated in three NSCLC cell lines (D). Bars represent the mean6the standard error of the mean (SEM) of three different experiments. **p,0.05 for PG-BE1 versus PG-LH7. *p,0.05 for PG-LH7 versus PAa. doi:10.1371/journal.pone.0058361.g003

Article Snippet: RANKL cDNA transfection Full-length human RANKL cDNA(RefSeq: NM_033012.2) was inserted into the eukaryotic vector pCMV6-XL5 (purchased from OriGene Technologies, Inc. Cat No: SC305532).

Techniques: Expressing, Western Blot

Journal: PloS one

Article Title: Differential expression of the RANKL/RANK/OPG system is associated with bone metastasis in human non-small cell lung cancer.

doi: 10.1371/journal.pone.0058361

Figure Lengend Snippet: Figure 4. Recombinant RANKL and RANKL cDNA stimulated PAa migration. Western blot analysis demonstrated higher RANKL protein expression in PAa-RANKL cells compared with PAa and PAa- Mock cells (A). Recombinant RANKL stimulated PAa cell migration, and the effect of RANKL administration was blocked by adding OPG to the culture medium in a dose-dependent manner. *p,0.05 for 300 ng/ml recombinant RANKL versus the control and 200 ng/ml OPG-treated samples (B). Increased migration of PAa-RANKL cells in vitro was demonstrated, and could be blocked by adding OPG to the culture medium. *p,0.05 for PAa-RANKL versus PAa, PAa-Mock and 200 ng/ml OPG-treated samples (C). Results are reported as the mean6the standard error of the mean (SEM) of triplicate assays. doi:10.1371/journal.pone.0058361.g004

Article Snippet: RANKL cDNA transfection Full-length human RANKL cDNA(RefSeq: NM_033012.2) was inserted into the eukaryotic vector pCMV6-XL5 (purchased from OriGene Technologies, Inc. Cat No: SC305532).

Techniques: Recombinant, Migration, Western Blot, Expressing, Control, In Vitro

Journal: PloS one

Article Title: Differential expression of the RANKL/RANK/OPG system is associated with bone metastasis in human non-small cell lung cancer.

doi: 10.1371/journal.pone.0058361

Figure Lengend Snippet: Figure 5. Plain radiographs and 18F-FDG micro PET/CT at 8- weeks in the study groups. A—Plain radiograph; B—micro CT(transverse view); C—micro PET/CT overlay(transverse view). Plain radiograph and micro PET/CT demonstrated increased bone destruc- tion(white arrows) and 18F-FDG uptake in group(PAa-RANKL) and group (PAa-RANKL+PBS) at 8-weeks following intratibial injection of tumor cells, whereas the increase was inhibited in group(PAa-RANKL+OPG). doi:10.1371/journal.pone.0058361.g005

Article Snippet: RANKL cDNA transfection Full-length human RANKL cDNA(RefSeq: NM_033012.2) was inserted into the eukaryotic vector pCMV6-XL5 (purchased from OriGene Technologies, Inc. Cat No: SC305532).

Techniques: Micro-PET, Micro-CT, Injection

Journal: PloS one

Article Title: Differential expression of the RANKL/RANK/OPG system is associated with bone metastasis in human non-small cell lung cancer.

doi: 10.1371/journal.pone.0058361

Figure Lengend Snippet: Figure 6. Protein staining and RANKL: OPG ratio in NSCLC primary lesions and bone metastases. Immunocytochemistry for RANKL, RANK, and OPG was performed in tissue sections from primary NSCLC lesions and bone metastases originating from NSCLC, and the staining intensities were evaluated (A). Next, the ratio of RANKL: OPG immunostaining density was calculated in primary NSCLC lesions and bone metastases originating from NSCLC (B). Results are expressed as the mean6the standard error of the mean (SEM) of three separate experiments. *p,0.05. doi:10.1371/journal.pone.0058361.g006

Article Snippet: RANKL cDNA transfection Full-length human RANKL cDNA(RefSeq: NM_033012.2) was inserted into the eukaryotic vector pCMV6-XL5 (purchased from OriGene Technologies, Inc. Cat No: SC305532).

Techniques: Staining, Immunocytochemistry, Immunostaining

Journal: Bone Research

Article Title: Osteomodulin downregulation is associated with osteoarthritis development

doi: 10.1038/s41413-023-00286-5

Figure Lengend Snippet: OMD inhibits osteoclastogenesis through its direct interaction with RANKL and balances bone remodeling. a Solid phase binding assay on the capture of RANKL by OMD. RANKL was coated on a plate followed by OMD addition. On the left: Binding assay with different concentrations of OMD (1 000 to 15.65 ng·mL −1 by serial 2X dilution), with 0.2 μg·mL −1 of coated RANKL (red curve) and negative control without RANKL (blue curve). On the right: Binding assay with different concentrations of coated RANKL (800 to 6.25 ng·mL −1 ) and 0.5 μg·mL −1 of given OMD (pink curve); negative control without OMD (purple curve). Wilcoxon test was performed with differences being considered significant at P values < 0.05 (* P < 0.05, ** P ≤ 0.01). b Assay of the effect of OMD on primary murine osteoclast culture. The peripheral blood mononuclear cells were collected from murine bone marrow and differentiated into osteoclasts with M-CSF and RANKL. Osteoclasts were counted after 4 days of differentiation following a TRAP staining. Each point represents a mouse, n = 7. The osteoclast count was represented in percentage of cells with the corresponding control set as 100%. Blue arrows point at osteoclasts. Scale bar = 50 µm. The data were plotted as a box plot showing all points. One-way ANOVA was performed with differences being considered significant at P values <0.05 (* P < 0.05). c Level of P1NP and TRAcP 5b measured in the serum of KO, WT and UP mice. The data were plotted as a box plot showing all points with n = 12 for the KO and WT, and n = 10 for the UP. One-way ANOVA was performed when the distribution was Gaussian and Kruskal–Wallis was performed when the distribution was not Gaussian with differences being considered significant at P values < 0.05 (* P < 0.05, ** P ≤ 0.01, **** P ≤ 0.000 1). d Schematic representation of the mechanism of OMD on osteoclastogenesis. Osteoblasts secrete RANKL which binds to the RANK receptor on the membrane of pre-osteoclasts to induce their differentiation into osteoclasts. In parallel, osteoblasts also secrete OMD which displays the ability to capture RANKL and would prevent its binding to RANK

Article Snippet: Human recombinant RANKL (OriGene, Germany) was bound for 2 h under constant agitation to Well-CoatedTM Nickel (G-Biosciences) previously washed with PBST.

Techniques: Binding Assay, Negative Control, Staining, Control, Membrane

Journal: Medicine

Article Title: Determination of diagnostic and predictive parameters for vertical mandibular invasion in patients with lower gingival squamous cell carcinoma: A retrospective study

doi: 10.1097/MD.0000000000032206

Figure Lengend Snippet: Immunohistochemical staining.

Article Snippet: IL-6 , Rockland Inc. , 1:2000 , Rabbit polyclonal antibody , • Inflammatory cytokines released by cancer cells. • Since stimulation by IL-6 and TNF-α induces bone-resorbing multinucleated giant cells, it is possible that some mechanisms of the bone-resorptive effects of IL-6 are not mediated by the RANK/RANKL pathway..

Techniques: Immunohistochemical staining, Staining, Binding Assay, Activity Assay, Expressing