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: MedComm

Article Title: HSD17B7 Counters Bone Loss in Estrogen Deficiency via Estrogen Receptor Stabilization and Mediates the Effect of Raloxifene

doi: 10.1002/mco2.70623

Figure Lengend Snippet: Deficiency of HSD17B7 induces estrogen‐deficiency osteoporosis by increasing the number of osteoclasts without affecting osteoblast function. (A) Representative micro‐CT reconstruction of cancellous bone, H&E staining (scale bars = 250 µm) and TRAP staining (scale bars = 40 µm) images of distal femoral metaphyses ( n = 4–5 animals/group). (B) Cancellous bone volume (BV/TV, %), trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular separation (Tb.Sp) were determined by a micro‐CT analysis. (C) Osteoclast numbers were analyzed in cancellous bone from the distal femur ( n = 7). (D) The RANKL/OPG ratio in serum was determined ( n = 3–4). (E) Six weeks after surgery, uterus weight was determined ( n = 6). (F) Representative TRAP staining images after osteoclastogenesis induction. Scale bars = 40 µm. TRAP‐positive multinucleate cells with three or more nuclei were counted as osteoclasts, and they were scored per field ( n = 5). (G) Calcein–Alizarin red label staining image and quantitative analysis of BFR (scale bars = 100 µm). (H, I) Representative images of ARS and ALP staining and quantitative analysis ( n = 4–5). The values presented are the mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 versus WT Sham, # p < 0.05 and ## p < 0.01, and ### p < 0.001 versus WT OVX.

Article Snippet: The primary antibodies used for Western blotting were HSD17B7, RANKL (1:1000 Santa Cruz Biotechnology), ERα (R&D Systems), HA, MYC, PLD1, phosphorylated PLD1, mTOR, phosphorylated mTOR, S6K, phosphorylated S6K, S6, phosphorylated S6, ubiquitin (1:1000 Cell Signaling Technology, Danvers, MA, USA), Col1a1, OPN, OPG, total OxPhos, NFATC1 (1:2000 Abcam), and GAPDH (1:2000 Cell Signaling Technology).

Techniques: Micro-CT, Staining

Journal: MedComm

Article Title: HSD17B7 Counters Bone Loss in Estrogen Deficiency via Estrogen Receptor Stabilization and Mediates the Effect of Raloxifene

doi: 10.1002/mco2.70623

Figure Lengend Snippet: Aggravation of OVX‐induced cancellous bone loss caused by HSD17B7 deficiency in preosteoclasts. (A) Representative micro‐CT reconstruction of cancellous bone, H&E staining (scale bars = 250 µm) and TRAP staining (scale bars = 40 µm) images of distal femoral metaphyses ( n = 4–5 animals/group). (B) Cancellous bone volume (BV/TV, %), trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular separation (Tb.Sp) were determined by a micro‐CT analysis. (C) Osteoclast numbers were analyzed in cancellous bone from the distal femur ( n = 7). (D) Western blot analysis of the protein levels of HSD17B7 and ERα in Sham/OVX mouse BMCs ( n = 3). (E) The RANKL/OPG ratio in serum was determined ( n = 5). (F) Six weeks after surgery, uterus weight was determined ( n = 4). (G) Representative images of immunofluorescence staining of HSD17B7 (green) and cathepsin K (red) in distal femoral metaphyses. (H) Representative images of immunofluorescence staining of HSD17B7 (green) and ERα (red) in CD11b + osteoclast cells (scale bars = 20 µm). The values presented are the mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 versus Control Sham, # p < 0.05 and ### p < 0.001 versus Control OVX.

Article Snippet: The primary antibodies used for Western blotting were HSD17B7, RANKL (1:1000 Santa Cruz Biotechnology), ERα (R&D Systems), HA, MYC, PLD1, phosphorylated PLD1, mTOR, phosphorylated mTOR, S6K, phosphorylated S6K, S6, phosphorylated S6, ubiquitin (1:1000 Cell Signaling Technology, Danvers, MA, USA), Col1a1, OPN, OPG, total OxPhos, NFATC1 (1:2000 Abcam), and GAPDH (1:2000 Cell Signaling Technology).

Techniques: Micro-CT, Staining, Western Blot, Immunofluorescence, Control

Journal: MedComm

Article Title: HSD17B7 Counters Bone Loss in Estrogen Deficiency via Estrogen Receptor Stabilization and Mediates the Effect of Raloxifene

doi: 10.1002/mco2.70623

Figure Lengend Snippet: Enhanced mitochondrial function in HSD17B7‐deficient preosteoclasts. (A) Heatmap of the RNA‐seq analysis of mitochondrial complex genes in CD11b + Control/cKO BMCs treated with M‐CSF (10 ng/mL) and RANKL (30 ng/mL). (B) qPCR for marker genes in CD11b + Control/cKO BMCs treated with M‐CSF (10 ng/mL) and RANKL (30 ng/mL; n = 4). (C) Representative Western blot results for the OxPhos complex, quantify the results ( n = 4), and quantitative qPCR analysis of mitochondrial DNA (mtDNA), using nuclear DNA (nDNA) as the standard ( n = 5). (D) OCR curves in CD11b + Control/cKO BMCs treated with M‐CSF (10 ng/mL), RANKL (30 ng/mL), oligomycin, FCCP, and rotenone/antimycin A ( n = 5). (E) CD11b + BMCs were stained using JC‐10 and observed via confocal microscopy. Representative images show merged polymeric (red) and monomeric (green) JC‐10 signals. The ratio of polymeric to monomeric JC‐10 was calculated. (F) Representative transmission electron micrographs of M‐CSF (10 ng/mL) and RANKL (30 ng/mL)‐treated CD11b + Control/cKO BMCs and quantitative analysis showing the numbers of mitochondria with normal and abnormal morphology. (G) Fragmentation of cellular DNA was detected by a TUNEL assay. Intracellular reactive oxygen species (ROS) were detected by H2DCFDA (green) (scale bars = 40 µm). The values presented are the mean ± SEM. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: The primary antibodies used for Western blotting were HSD17B7, RANKL (1:1000 Santa Cruz Biotechnology), ERα (R&D Systems), HA, MYC, PLD1, phosphorylated PLD1, mTOR, phosphorylated mTOR, S6K, phosphorylated S6K, S6, phosphorylated S6, ubiquitin (1:1000 Cell Signaling Technology, Danvers, MA, USA), Col1a1, OPN, OPG, total OxPhos, NFATC1 (1:2000 Abcam), and GAPDH (1:2000 Cell Signaling Technology).

Techniques: RNA Sequencing, Control, Marker, Western Blot, Staining, Confocal Microscopy, Transmission Assay, TUNEL Assay

Journal: MedComm

Article Title: HSD17B7 Counters Bone Loss in Estrogen Deficiency via Estrogen Receptor Stabilization and Mediates the Effect of Raloxifene

doi: 10.1002/mco2.70623

Figure Lengend Snippet: Enhanced oxidative capacity of HSD17B7 cKO mice via the PLD1–mTOR signaling pathway. (A) Volcano plot showing the log2 fold‐difference in M‐CSF (10 ng/mL) and RANKL (30 ng/mL)‐treated CD11b + Control/cKO BMCs, assessed using a StringTie analysis of RNA‐seq data. Red and blue dots represent upregulated and downregulated DEGs, with > twofold change and p < 0.05, respectively. Each sample was analyzed in triplicate. (B) Bubble plots show significant changes in the KEGG analysis. (C) Representative Western blot analysis of the PLD1–mTOR signaling pathway and the results were quantified ( n = 4). (D) PDL1 activity from preosteoclasts. (E–G) CD11b + cKO BMCs were transfected with ERα or empty vector (EV) by electroporation for 24 h. (E) Representative the expression patterns of PLD1–mTOR signaling pathway were determined via Western blot and quantify the results ( n = 4). (F) Representative TRAP staining after osteoclastogenesis induction. Scale bars = 100 µm. TRAP‐positive multinucleate cells with three or more nuclei were counted as osteoclasts and were scored per field ( n = 8). (G) PDL1 activity from preosteoclasts. (H–K) CD11b + Control/cKO BMCs were treated with VU0359595 (1 µM) for 24 h. (H) Representative the expression patterns of the PLD1–mTOR signaling pathway and OxPhos complex were determined via Western blotting. (I) qPCR for marker genes after treatment with VU0359595. (J) Representative TRAP staining images after osteoclastogenesis induction. Scale bars = 200 µm. TRAP‐positive multinucleate cells with three or more nuclei were counted as osteoclasts, and they were scored per field ( n = 5). (K) OCR curves in VU0359595‐treated CD11b + Control/cKO BMCs also treated with oligomycin, FCCP, and rotenone/antimycin A ( n = 3). The values presented are the mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 versus Control, # p < 0.05, ## p < 0.01, ### p < 0.001 versus cKO or EV‐treated cKO.

Article Snippet: The primary antibodies used for Western blotting were HSD17B7, RANKL (1:1000 Santa Cruz Biotechnology), ERα (R&D Systems), HA, MYC, PLD1, phosphorylated PLD1, mTOR, phosphorylated mTOR, S6K, phosphorylated S6K, S6, phosphorylated S6, ubiquitin (1:1000 Cell Signaling Technology, Danvers, MA, USA), Col1a1, OPN, OPG, total OxPhos, NFATC1 (1:2000 Abcam), and GAPDH (1:2000 Cell Signaling Technology).

Techniques: Control, RNA Sequencing, Western Blot, Activity Assay, Transfection, Plasmid Preparation, Electroporation, Expressing, Staining, Marker

Journal: MedComm

Article Title: HSD17B7 Counters Bone Loss in Estrogen Deficiency via Estrogen Receptor Stabilization and Mediates the Effect of Raloxifene

doi: 10.1002/mco2.70623

Figure Lengend Snippet: Attenuation of HSD17B7–ERα expression in severely osteoporotic patients. Human CD11b + BMCs were treated with M‐CSF (25 ng/mL) and RANKL (50 ng/mL) for 7 days. (A) Scatterplots of HSD17B7 expression and various bone parameters. The coefficient of determination was used to compare the association between HSD17B7 expression and the bone parameters, with red and blue representing negative and positive correlation, respectively ( n = 16). (B) Representative H&E staining (scale bars = 250 µm), TRAP staining (scale bars = 40 µm) and immunofluorescence staining (scale bars = 40 µm) images of osteoclasts in control subjects and osteoporotic patients. (C) Human CD11b + BMCs were treated with M‐CSF and RANKL for 7 days, and the expression of HSD17B7 and ERα proteins was analyzed using Western blotting. (D) Clinical parameters of control subject and osteoporotic patients. The values presented are the mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 versus control. Control: T‐score >‐ 1, Osteoporosis: T‐score ← 2.5, Severe osteoporosis: T‐score ← 2.5 with bone fracture.

Article Snippet: The primary antibodies used for Western blotting were HSD17B7, RANKL (1:1000 Santa Cruz Biotechnology), ERα (R&D Systems), HA, MYC, PLD1, phosphorylated PLD1, mTOR, phosphorylated mTOR, S6K, phosphorylated S6K, S6, phosphorylated S6, ubiquitin (1:1000 Cell Signaling Technology, Danvers, MA, USA), Col1a1, OPN, OPG, total OxPhos, NFATC1 (1:2000 Abcam), and GAPDH (1:2000 Cell Signaling Technology).

Techniques: Expressing, Staining, Immunofluorescence, Control, Western Blot

Journal: MedComm

Article Title: HSD17B7 Counters Bone Loss in Estrogen Deficiency via Estrogen Receptor Stabilization and Mediates the Effect of Raloxifene

doi: 10.1002/mco2.70623

Figure Lengend Snippet: The anti‐bone‐resorption effect of raloxifene in estrogen‐deficient osteoporosis is mediated by HSD17B7. (A) Control and cKO mice were treated with raloxifene (RAL) (0.5 mg/kg) via subcutaneous injection 5 days/week for 5 weeks during the OVX program. Representative micro‐CT reconstruction of cancellous bone, H&E staining (scale bars = 250 µm), and TRAP staining (scale bars = 40 µm) images of distal femoral metaphyses ( n = 4–5 animals/group). (B) Cancellous bone volume (BV/TV, %), trabecular thickness (Tb.Th), trabecular number (Tb.N), trabecular separation (Tb.Sp), and uterus weight were determined by a micro‐CT analysis. (C) Representative TRAP staining images after osteoclastogenesis induction. Scale bars = 100 µm ( n = 5). (D) OCR curves from CD11b + Control/cKO BMCs treated with RAL (3 µM), M‐CSF (10 ng/mL), RANKL (30 ng/mL), oligomycin, FCCP, and rotenone/antimycin A ( n = 3–4). (E) Calculation of the ratio of polymeric to monomeric JC‐10. (F) CD11b + Control/cKO BMCs were treated with RAL (3 µM) for 24 h, and representative the expression patterns of the PLD1–mTOR signaling pathway were determined via Western blotting and the results were quantified ( n = 3). (G) PDL1 activity from preosteoclasts. (H) Co‐IP and immunoblot analyses of CD11b + Control/cKO BMCs treated with RAL. * p < 0.05, ** p < 0.01 versus Control Sham or Control DMSO, # p < 0.05, ## p < 0.01, ### p < 0.001 versus Control OVX, cKO OVX, or cKO DMSO.

Article Snippet: The primary antibodies used for Western blotting were HSD17B7, RANKL (1:1000 Santa Cruz Biotechnology), ERα (R&D Systems), HA, MYC, PLD1, phosphorylated PLD1, mTOR, phosphorylated mTOR, S6K, phosphorylated S6K, S6, phosphorylated S6, ubiquitin (1:1000 Cell Signaling Technology, Danvers, MA, USA), Col1a1, OPN, OPG, total OxPhos, NFATC1 (1:2000 Abcam), and GAPDH (1:2000 Cell Signaling Technology).

Techniques: Control, Injection, Micro-CT, Staining, Expressing, Western Blot, Activity Assay, Co-Immunoprecipitation Assay

Journal: Cancer Research

Article Title: Therapeutic Relevance of Osteoprotegerin Gene Therapy in Osteosarcoma: Blockade of the Vicious Cycle between Tumor Cell Proliferation and Bone Resorption

doi: 10.1158/0008-5472.can-06-4130

Figure Lengend Snippet: Figure 5. OPG-Fc does not bind to osteosarcoma cells nor affect osteosarcoma cell proliferation and apoptosis. Murine osteosarcoma POS-1 cells were incubated with FITC-labeled murine OPG-Fc and analyzed by FACS (A). Human fetal 2.93 cells overexpressing the murine form of full-length RANKL (293 RL 2.0) were used as positive controls. The effects were assessed on POS-1 cell proliferation using an XTT assay as described in Materials and Methods. POS-1 cells were treated for 12 to 72 h with increasing concentrations of murine OPG-Fc (10, 50, and 100 ng/mL) that exhibits the same sequence as the mOPGD transgene used for in vivo experiments (B). To determine the OPG influence on tumor cell apoptosis, the caspase-3 activity was analyzed in the cell lysate of POS-1 cells treated with 100 ng/mL mOPG-Fc for 24, 48, and 72 h (C), CT+, staurosporin (1 Amol/L, 6 h) was used as positive control; CT, POS-1 cells alone. Parallel experiments were done on cell cycle distribution in the absence or the presence of 100 ng/mL mOPG-Fc during 24, 48, and 72 h (D; because no effect could be detected, only the results obtained after 72 h of incubation are shown).

Article Snippet: RANKL expression was also compared at the tissue level between control mice (bone, muscle) and osteosarcoma-bearing mice (tumor) by immunohistochemistry using a polyclonal rabbit anti-RANKL antibody (RANKL-N19; 1/100, Santa Cruz).

Techniques: Incubation, Labeling, XTT Assay, Sequencing, In Vivo, Activity Assay, Positive Control

Journal: Cancer Research

Article Title: Therapeutic Relevance of Osteoprotegerin Gene Therapy in Osteosarcoma: Blockade of the Vicious Cycle between Tumor Cell Proliferation and Bone Resorption

doi: 10.1158/0008-5472.can-06-4130

Figure Lengend Snippet: Figure 6. Comparison of tumor cell apoptosis, osteoclast number, and RANKL expression between POS-1 osteosarcoma bearing mice treated or not with p-mOPGD. A, tumor cell apoptosis was investigated in vivo by TUNEL staining and compared between POS-1 mice treated or not with OPG (POS-1/p-mOPGD). Original magnification, 400. B, the effect of p-mOPGD on osteoclast number was studied by TRACP 5b activity in the serum of POS-1 osteosarcoma bearing mice. C, RANKL production was measured by ELISA test in the serum of POS-1 osteosarcoma-bearing mice treated or not with OPG (POS-1/p-mOPGD) as compared with control mice (CT). D, localization of RANKL expression was investigated by immunohistochemistry in the tumor microenvironment of POS-1 osteosarcoma-bearing mice treated or not with OPG (POS-1 and POS-1/p-mOPGD). Original magnification, 640.

Article Snippet: RANKL expression was also compared at the tissue level between control mice (bone, muscle) and osteosarcoma-bearing mice (tumor) by immunohistochemistry using a polyclonal rabbit anti-RANKL antibody (RANKL-N19; 1/100, Santa Cruz).

Techniques: Comparison, Expressing, In Vivo, TUNEL Assay, Staining, Activity Assay, Enzyme-linked Immunosorbent Assay, Control, Immunohistochemistry

Journal: Immunity, Inflammation and Disease

Article Title: RANKL Attenuates Sepsis‐Associated Acute Lung Injury Through the OPG/RANKL/RANK/TLR4 Pathway

doi: 10.1002/iid3.70356

Figure Lengend Snippet: Comparison of mRNA expression levels of TLR4, RANK, RANKL, and OPG in lung tissues of mice in each group. The mRNA levels were detected with real‐time quantitative PCR. (A) Level of RANKL mRNA. (B) Level of OPG mRNA. (C) Level of RANK mRNA. (D) Level of TLR4 mRNA. *** p < 0.001, **** p < 0.0001.

Article Snippet: After that, the pre‐diluted primary antibodies against OPG (cat# DF6824; Affinity Bioscience), RANKL (cat# 23408‐1‐AP; Proteintech Group Inc.), RANK (cat# ab222215; Abcam), TLR4 (cat# 19811‐1‐AP; Proteintech Group Inc.), and β‐actin (cat# TDY051; Tiandeyue Biotechnology Co. Ltd) were added and probed overnight at 4°C.

Techniques: Comparison, Expressing, Real-time Polymerase Chain Reaction

Journal: Immunity, Inflammation and Disease

Article Title: RANKL Attenuates Sepsis‐Associated Acute Lung Injury Through the OPG/RANKL/RANK/TLR4 Pathway

doi: 10.1002/iid3.70356

Figure Lengend Snippet: Comparison of protein expression levels of TLR4, RANK, RANKL, and OPG in lung tissues of mice in each group. The protein levels were detected with Western blot analysis. (A) Representative Western blot results. (B) The relative protein level of RANKL. (C) The relative protein level of OPG. (D) The relative protein level of RANK. (E) The relative protein level of TLR4. * p < 0.05, **** p < 0.0001.

Article Snippet: After that, the pre‐diluted primary antibodies against OPG (cat# DF6824; Affinity Bioscience), RANKL (cat# 23408‐1‐AP; Proteintech Group Inc.), RANK (cat# ab222215; Abcam), TLR4 (cat# 19811‐1‐AP; Proteintech Group Inc.), and β‐actin (cat# TDY051; Tiandeyue Biotechnology Co. Ltd) were added and probed overnight at 4°C.

Techniques: Comparison, Expressing, Western Blot

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: 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: Fig. 8 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: Total RNA was extracted from osteoblast cultures, with RNA quality indicator scores (RIN) of 9.3, and RNA-seq for differential gene expression analyses was performed with a false discovery rate (FDR) of 0.01 to assess the statistical significance as described in ref. 63 Solid phase binding assay 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