Journal: The FASEB Journal

Article Title: Amyloid β peptide promotes bone formation by regulating Wnt/β‐catenin signaling and the OPG/RANKL/RANK system

doi: 10.1096/fj.201901550r

Figure Lengend Snippet: FIGURE 5 Aβ was a negative regulator of osteoclast differentiation in the co-culture system. A, ALP staining in co-cultured MC3T3-E1 with and without Aβ. B, TRAP staining of osteoclasts and the number of TRAP (+) multinucleated osteoclasts generated from co-cultured RAW264.7 cells with and without Aβ. C, qPCR analysis of Wnt3a, OPG and RANKL expression at 5 days after osteoblast differentiation of co-cultured MC3T3-E1. D, ELISA analysis of OPG expression at day 5 in the co-culture cell media with and without XAV-939 and/or Wnt3a. (a, P < .05 vs the control group; b, P < .05 vs the 5 μM Aβ group) E, Western blot analysis of TCF and OPG in co-cultured MC3T3-E1 with or without Wnt inhibitor XAV-939 or Wnt agonist Wnt3a. F, Western blot analysis of IκB-α and NFATc1 in co-cultured RAW264.7 cells with or without Aβ, or XAV-939. Data are represented as the mean ± SD of three independent experiments. *P < .05, **P < .01 vs Control group. For (D) (E) (F), aP < .05, vs the control group; bP < .05, vs the Aβ, group; cP < .05, vs the Aβ+XAV-939, group; cP < .05, vs the Aβ+XAV-939, group; OPG, osteoprotegerin; RANKL, Receptor Activator for Nuclear Factor-κB Ligand

Article Snippet: Medium levels of OPG and RANKL were measured using mouse OPG and RANKL ELISA kit (Elabscience Biotechnology, China).

Techniques: Co-Culture Assay, Staining, Cell Culture, Generated, Expressing, Enzyme-linked Immunosorbent Assay, Control, Western Blot

Journal: Cancer Research Communications

Article Title: OPG-Producing B Cells and RANKL-Expressing T Cells Define Immune Signatures Predictive of Bone Metastases in Breast Cancer

doi: 10.1158/2767-9764.CRC-25-0696

Figure Lengend Snippet: Silencing of OPG in 67NR-primed CD19 + B cells abrogates their regulatory activity and restores 4T1 osteolytic and metastatic phenotypes in vivo . A, Experimental scheme: CD19 + B cells were isolated from the iliac BM of BALB/c mice bearing 67NR tumors on day 11 after implantation. Cells were subjected to gene silencing using lentiviral vectors carrying shRNA targeting Tnfrsf11b (OPG; B 67NR sh OPG) or a nontargeting scrambled control (B 67NR sh scr). Naive mice were used as experimental controls (Nv). B, OPG levels in culture supernatants from CD19 + B cells were assessed by ELISA. C, Experimental setup for in vivo analysis: OPG-silenced (sh OPG) or control-transduced (sh scr) CD19 + B cells were adoptively transferred together with CD3 + T cells from 4T1 tumor-bearing mice into immunocompetent BALB/c mice implanted with 1 × 10 5 4T1 tumor cells. D, Primary tumor growth was monitored over time. E, Representative images of tumor-bearing mice after adoptive B cell transfer. Photographs were taken at the experimental endpoint to illustrate macroscopic differences in tumor burden among groups. F, Quantification of RANKL levels by ELISA in supernatants of in vitro restimulated LNs and BM cells. G, Metastatic burden in draining LNs and BM was assessed by clonogenic assays. Data are presented as mean ± SD from two independent experiments ( n = 5 mice/group). Statistical comparisons between more than two groups were performed using one-way ANOVA followed by Tukey post hoc test for pairwise comparisons when data were normally distributed. Statistical significance was considered when P < 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: To silence OPG expression in BM–derived CD19 + B cells from 67NR tumor–bearing BALB/c mice (day 11), cells were transfected with a specific murine short hairpin (sh) RNA (shRNA) plasmid targeting OPG [sc-40153-SH, Santa Cruz Biotechnology] or with a nontargeting control shRNA plasmid (sc-108060).

Techniques: Activity Assay, In Vivo, Isolation, shRNA, Control, Enzyme-linked Immunosorbent Assay, In Vitro

Journal: Cells

Article Title: Osteoprotegerin (OPG) Upregulation Activates Breast Stromal Fibroblasts and Enhances Their Pro-Carcinogenic Effects through the STAT3/IL-6 Signaling

doi: 10.3390/cells11213369

Figure Lengend Snippet: Breast cancer cells and IL-6 upregulate OPG in CAFs through the IL-6/STAT3 pathway. ( A ) Whole cell lysates were prepared from the indicated cells and were used for immunoblotting analysis. The numbers below the bands represent OPG relative expression after correction against the internal control GAPDH. ( B ) Serum-free conditioned media from CAF-64, CAF-87 (CAFs), TCF-64, TCF-87 (TCFs) and NBF-1, NBF-6, NBF-20, NBF-25 (NBFs) were collected after 24 h of incubation with SFM and the levels of the OPG protein were determined by ELISA. Error bars represent mean ± SEM, * p < 0.05; **** p < 0.0001 by Ordinary one-way ANOVA. ( C ) NBF6 cells were cultured either in SFM or in MDA-MB-231 SFCM (MDA-SFCM) for 24 h, and then whole cell lysates were prepared for immunoblotting using specific antibodies for the indicated proteins. ( D ) NBF6 cells were cultured either in SFM or SFM containing 3.5 ng/mL of the rhIL-6 protein for 24 h. Whole cell lysates were prepared for immunoblotting analysis using specific antibodies for the indicated proteins. All values were determined by densitometry relative to GAPDH and presented as fold change relative to the respective controls. ( E , F ) NBF6 cells were transfected with STAT3 siRNA (STAT3si) or a scrambled sequence (Ctrl). Ctrl and STAT3si cells were either not treated ( E ) or exposed to MDA-MB-231 SFCM (MDA-SFCM) for 24 h ( F ). Cell lysates were then prepared for immunoblotting using specific antibodies for the indicated proteins. The numbers below the bands represent fold change relative to the control (SFM) after correction against the internal control GAPDH. The level of the phosphorylated-STAT3 was normalized against the non-phosphorylated form of the protein.

Article Snippet: STAT3 siRNA was obtained from Qiagen, OPG siRNA and control siRNA were obtained from (Santa Cruz, Santa Cruz, CA, USA).

Techniques: Western Blot, Expressing, Control, Incubation, Enzyme-linked Immunosorbent Assay, Cell Culture, Transfection, Sequencing

Journal: Cells

Article Title: Osteoprotegerin (OPG) Upregulation Activates Breast Stromal Fibroblasts and Enhances Their Pro-Carcinogenic Effects through the STAT3/IL-6 Signaling

doi: 10.3390/cells11213369

Figure Lengend Snippet: OPG downregulation suppresses active CAFs. CAF-64 and CAF-87 cells were transfected with specific OPG-siRNA (CAF64-OPGsi and CAF87-OPGsi) and a scrambled sequence was used as a control (CAF64-Ctl and CAF87-Ctl), respectively. ( A ) Total RNA was extracted from the indicated cells, and the mRNA levels of the indicated genes were assessed by qRT-PCR using specific primers for the indicated genes. Error bars represent mean ± S.D. * p < 0.05, ** p < 0.005. ( B ) Whole cell lysates were prepared from the indicated cells, and then were used for immunoblotting analysis using specific antibodies against the indicated proteins. The numbers below the bands indicate band intensity normalized to GAPDH, while phospho-proteins were further normalized to the total protein and presented as fold change as compared to the control. ( C ) Exponentially growing cells were seeded in E-plate for proliferation and CIM-plate for migration, and cell proliferation/migration were assessed using the Real-Time Cell Analyzer-Dual Plate (RTCA-DP) xCELLigence System. Data are representative of different experiments performed in triplicate. ( D ) SFCM from the indicated cells were collected after 24 h and the levels of the indicated proteins were determined by ELISA and presented in the respective histograms. Error bars indicate mean ± SEM ( n = 3). * p < 0.05, ** p < 0.005.

Article Snippet: STAT3 siRNA was obtained from Qiagen, OPG siRNA and control siRNA were obtained from (Santa Cruz, Santa Cruz, CA, USA).

Techniques: Transfection, Sequencing, Control, Quantitative RT-PCR, Western Blot, Migration, Enzyme-linked Immunosorbent Assay

Journal: Proteomics

Article Title: Proteome-wide changes induced by the Hsp90 inhibitor, geldanamycin in anaplastic large cell lymphoma cells.

doi: 10.1002/pmic.200700108

Figure Lengend Snippet: Figure 6. Immunoblot validation of differentially expressed pro- teins as identified by LC-MS/MS. Total cell lysate was extracted from control and GA-treated cells after 12 h and proteins sepa- rated by SDS-PAGE. Immunoblots were probed with anti-USP9 (1:1000), anti-GAP1 (1:300), anti-PCLN (1:300), anti-STCN (1:300), anti-tankyrase (1:1000), anti-SOCS-4 (1:1000), anti-NEMO (1:1000), anti-OPG (1:500), and anti-actin (1:500). Immunoblots were then incubated at room temperature for 1 h followed by probing with respective HRP-conjugated secondary antibodies and visualized by chemiluminescence. The left panels depict the overexpressed proteins as identified by LC-MS/MS in control and cells exposed to 10 mM GA. The right panels depict the under- expressed proteins as identified in the same samples. Both cICAT- and densitometry fold-change is listed below the 10 mM GA lanes. Actin served as a loading control.

Article Snippet: The following antibodies were used for immunoblot analysis: mouse polyclonal antibodies against PARP, p21, and p27; goat polyclonal antibodies against ras GTPase-activating protein 3 (RARS3), paracellin-1 (PCLN1), and stanniocalcin-1 (STCN1); and rabbit polyclonal antibody against actin from Santa Cruz Biotechnology (Santa Cruz, CA); mouse polyclonal antibodies against Hsp90 and NEMO (Becton Dickinson Pharmingen, San Diego); rabbit polyclonal antibodies against phospho-ALK and ALK (Cell Signaling, Beverly, MA); rabbit polyclonal antibody against ubiquitin-specific protease 9 (USP9) (Abgent, San Diego); mouse polyclonal antibodies against tankyrase (TNKS) and osteoprotegerin (OPG) and rabbit polyclonal antibodies against suppressor of cytokine signaling 4 (SOCS4; Acris Antibodies, Hiddenhausen, Germany).

Techniques: Western Blot, Biomarker Discovery, Liquid Chromatography with Mass Spectroscopy, Control, SDS Page, Incubation

Journal: Frontiers in Pharmacology

Article Title: Meclizine Prevents Ovariectomy-Induced Bone Loss and Inhibits Osteoclastogenesis Partially by Upregulating PXR

doi: 10.3389/fphar.2017.00693

Figure Lengend Snippet: Meclizine reduces OVX-induced osteoclast formation and decreases the serum levels of CTX-I, OPG and RANKL. (A) The paraffin-embedded bone sections of distal femurs were stained for H&E and TRAP. (B) The osteoclast number/bone surface was quantified. Data represent as mean ± SD. n = 10. ∗∗ P < 0.01. (C) Serum levels of CTX-I, OPG, and RANKL were determined by ELISA. Data are presented as mean ± SD. n = 10. ∗∗ P < 0.01.

Article Snippet: Serum RANKL and OPG levels were evaluated by mouse RANKL and OPG ELISA kit (Boster).

Techniques: Staining, Enzyme-linked Immunosorbent Assay

Journal: Molecular Medicine Reports

Article Title: Effects of zinc carnosine on bone loss in mice with diabetic osteoporosis

doi: 10.3892/mmr.2025.13723

Figure Lengend Snippet: COL-I, OCN, OPG and RANKL protein expression levels in each group after 8 weeks of intervention. (A) COL-I, OCN, OPG and RANKL protein expression level of each treatment group. Average optical density of (B) COL-I, (C) OCN, (D) OPG and (E) RANKL in each group. *P<0.05 vs. Ctrl group; # P<0.05 vs. T2DM model group. COL-I, type I collagen; OCN, osteocalcin; OPG, osteoprotegerin; RANKL, receptor activator of nuclear factor-κB ligand; Ctrl, control; T2DM, type 2 diabetes mellitus; ZnC, zinc carnosine; AOD, average optical density value.

Article Snippet: The primary antibodies used and their dilution ratios are as follows: Type I collagen (COL-I; 1:200; cat. no. AF7001; Affinity Biosciences), osteocalcin (OCN, 1:200; cat. no. 16157-1-AP; Proteintech Group, Inc.) and OPG (1:200; cat. no 31766-1-AP; Proteintech Group, Inc.).

Techniques: Expressing, Control

Journal: Molecular Medicine Reports

Article Title: Effects of zinc carnosine on bone loss in mice with diabetic osteoporosis

doi: 10.3892/mmr.2025.13723

Figure Lengend Snippet: mRNA expression levels of bone metabolism-related factors. *P<0.05 and ***P<0.01 vs. Ctrl group; ## P<0.01 vs. T2DM model group. (A) Ctrl, control; (B) T2DM, type 2 diabetes mellitus; (C) ZnC, zinc carnosine. OCN, osteocalcin; OPG, osteoprotegerin; RANKL, receptor activator of nuclear factor-κB ligand.

Article Snippet: The primary antibodies used and their dilution ratios are as follows: Type I collagen (COL-I; 1:200; cat. no. AF7001; Affinity Biosciences), osteocalcin (OCN, 1:200; cat. no. 16157-1-AP; Proteintech Group, Inc.) and OPG (1:200; cat. no 31766-1-AP; Proteintech Group, Inc.).

Techniques: Expressing, Control