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: Journal of Dental Sciences

Article Title: T cell and periosteum cooperation in osteoclastogenesis induced by lipopolysaccharide injection in transplanted mouse tibia

doi: 10.1016/j.jds.2018.03.001

Figure Lengend Snippet: (A) The information of animal experimental schedule (date), groups, and sample size(n). (B) Schema of the tibial surface for histometric analysis. The number of TRAP-positive mononuclear and multinuclear cells, RANKL-positive cells, and CD3-positive cells was counted in a rectangular area (50 × 1000 μm) on the center of the tibia.

Article Snippet: Sections were then immersed in rabbit anti-mouse RANKL polyclonal antibody (1:200; Acris Antibodies, Rockville, MD, USA) and CD3 polyclonal antibody (1:200; Abcam, Cambridge, UK) at 4 °C overnight.

Techniques:

Journal: Journal of Dental Sciences

Article Title: T cell and periosteum cooperation in osteoclastogenesis induced by lipopolysaccharide injection in transplanted mouse tibia

doi: 10.1016/j.jds.2018.03.001

Figure Lengend Snippet: Immunohistological findings (A–D), and the number of RANKL-positive cells (E). (A–D) Arrowheads indicate RANKL-positive cells. (E) The number of RANKL-positive cells was highest in the WT (+) LPS group. Scale bars represent 25 μm. Bars represent mean ± SD. * P < 0.01 vs. WT (+) LPS group.

Article Snippet: Sections were then immersed in rabbit anti-mouse RANKL polyclonal antibody (1:200; Acris Antibodies, Rockville, MD, USA) and CD3 polyclonal antibody (1:200; Abcam, Cambridge, UK) at 4 °C overnight.

Techniques:

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Periodontal ligament cells under mechanical stress induce osteoclastogenesis by receptor activator of nuclear factor kappaB ligand up-regulation via prostaglandin E2 synthesis.

doi: 10.1359/jbmr.2002.17.2.210

Figure Lengend Snippet: FIG. 3. Compressive force up- regulated RANKL mRNA ex- pression in PDL cells. (A) RT- PCR analysis of PDL cells. PDL cells were loaded at different compressive forces (0, 0.5, 1.0, 2.0, or 3.0 g/cm2) for 24 h or at a constant compressive force (2 g/cm2) for 0, 0.5, 1.5, 6, 24, or 48 h. The results of one of four representative independent exper- iments are shown. (B) Densitom- etry analysis. The results are ex- pressed as the mean ratio to actin expression of four independent experiments.

Article Snippet: The primary antibody used in this experiment was goat polyclonal anti-RANKL antibody (sc7627; Santa Cruz Biotechnology, Inc.).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Periodontal ligament cells under mechanical stress induce osteoclastogenesis by receptor activator of nuclear factor kappaB ligand up-regulation via prostaglandin E2 synthesis.

doi: 10.1359/jbmr.2002.17.2.210

Figure Lengend Snippet: FIG. 5. Exogenous PGE2 treat- ment increased RANKL mRNA expression in PDL cells. (A) RT- PCR analysis of PDL cells. PDL cells were stimulated with PGE2 (0, 109, 108, or 107 M) for 24 h. PDL cells were stimulated with PGE2 (107 M) for the indi- cated times. The results of one representative independent exper- iment out of four are shown. (B) Densitometry analysis. The re- sults are expressed as the mean ratio to actin expression of four independent experiments.

Article Snippet: The primary antibody used in this experiment was goat polyclonal anti-RANKL antibody (sc7627; Santa Cruz Biotechnology, Inc.).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Periodontal ligament cells under mechanical stress induce osteoclastogenesis by receptor activator of nuclear factor kappaB ligand up-regulation via prostaglandin E2 synthesis.

doi: 10.1359/jbmr.2002.17.2.210

Figure Lengend Snippet: FIG. 6. The expression of RANKL protein increased with compres- sive force or PGE2 treatment. PDL cells were exposed to compressive force or PGE2 for 2 days or 4 days and then whole cell lysate from the cells was subjected to Western blot analysis with antibody against RANKL. Lanes 1 and 2 are controls. Lane 3 is the 2-day PGE2 treatment. Lane 4 is the 4-day PGE2 treatment. Lane 5 is the 2-day compressive force treatment. Lane 6 is the 4-day compressive force treatment. The results of one of three representative independent ex- periments are shown.

Article Snippet: The primary antibody used in this experiment was goat polyclonal anti-RANKL antibody (sc7627; Santa Cruz Biotechnology, Inc.).

Techniques: Expressing, Western Blot

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Periodontal ligament cells under mechanical stress induce osteoclastogenesis by receptor activator of nuclear factor kappaB ligand up-regulation via prostaglandin E2 synthesis.

doi: 10.1359/jbmr.2002.17.2.210

Figure Lengend Snippet: FIG. 8. Indomethacin inhibited RANKL mRNA up-regulation stimulated by compressive force. (A) RT-PCR analysis of PDL cells. PDL cells were stimulated with compressive force with or without indomethacin. Lane a contains control PDL cells without indomethacin. Lane b contains compressive force-loaded PDL cells without indomethacin. Lane c contains control PDL cells with indomethacin. Lane d contains compressive force-loaded PDL cells with indo- methacin. The results of one of four representa- tive independent experiments are shown. (B) Densitometry analysis. The results are expressed as the mean ratio to actin expression of four independent experiments.

Article Snippet: The primary antibody used in this experiment was goat polyclonal anti-RANKL antibody (sc7627; Santa Cruz Biotechnology, Inc.).

Techniques: Reverse Transcription Polymerase Chain Reaction, Control, Expressing

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Periodontal ligament cells under mechanical stress induce osteoclastogenesis by receptor activator of nuclear factor kappaB ligand up-regulation via prostaglandin E2 synthesis.

doi: 10.1359/jbmr.2002.17.2.210

Figure Lengend Snippet: FIG. 9. A model illustrating the mechanism by which compressive force-loaded PDL cells induce osteoclastogenesis. First, compressive force induces COX-2 expression, promoting PGE2 production. Second, increasing PGE2 affects PDL cells via an autocrine mechanism, result- ing in the up-regulation of RANKL expression in PDL cells. RANKL expression in PDL cells was up-regulated by compressive force, whereas OPG expression in PDL cells was not affected by compressive force. Finally, up-regulated RANKL expression induces osteoclasto- genesis from osteoclast progenitors in compressive force-loaded PDL cells.

Article Snippet: The primary antibody used in this experiment was goat polyclonal anti-RANKL antibody (sc7627; Santa Cruz Biotechnology, Inc.).

Techniques: Expressing

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: Journal of orofacial orthopedics = Fortschritte der Kieferorthopadie : Organ/official journal Deutsche Gesellschaft fur Kieferorthopadie

Article Title: Impact of phosphorylation of heat shock protein 27 on the expression profile of periodontal ligament fibroblasts during mechanical strain.

doi: 10.1007/s00056-022-00391-w

Figure Lengend Snippet: Fig. 2 Cell number (a) and lactate dehydrogenase (LDH) release (b) after exposure of human periodontal ligament fibroblasts (PDLF) to 6g/cm2 for 6h. Expression of COL1A2 mRNA (c), IL1B mRNA (d), IL6 mRNA (e), PTGS2 protein (f), RANKL protein (g) and OPG pro- tein (h) as well as determination of TRAP+ cells in a coculture model using THP1 macrophages (i) after exposure of PDLF to 6g/cm2 for 6h. Statistics: Mann–Whitney U test; *P< 0.05; **P< 0.01; ***P< 0.001 Abb. 2 Zellzahl (a) und LDH(Laktatdehydrogenase)-Freisetzung (b) nach 6-stündiger Exposition von PDLF (parodontale Ligamentfi- broblasten) mit 6g/cm2. Expression von COL1A2 mRNA (c), IL1B mRNA (d), IL6 mRNA (e), PTGS2-Protein (f), RANKL-Protein (g) und OPG-Protein (h) sowie Bestimmung von TRAP+-Zellen in ei- nem Kokulturmodell mit THP1-Makrophagen (i) nach 6-stündiger Exposition von PDLF mit 6g/cm2. Statistik: Mann-Whitney-U-Test; *p< 0,05; **p< 0,01; ***p< 0,001

Article Snippet: This was followed by incubation with the diluted primary antibodies (HSP27 1:3000 (AF1580, R&D Systems, Minneapolis, MN, USA); pHSP27 1:500 (AF2314, R&D Systems, Minneapolis, MN, USA), PTGS2 1:2500 (PA5-1817, Thermo Fisher Scientific, Waltham, MA, USA), RANKL 1:2000 (TA306362, OriGene, Rockville, MD, USA); ACTIN 1:1000 (E1C602, ABIN274248, Antibody-online, Aachen, Germany)).

Techniques: Expressing, MANN-WHITNEY