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Fig. 7 Fer-1 inhibits osteoporosis of OVX mice. (A) Reconstruction of bone trabecular of distal femurs by Micro-CT of each group. Scale bar = 200 μm. (B-F) Quantitative micro-CT analysis of bone mineral density (BMD), bone volume/total volume ratio (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular separation (Tb.Sp). (G) HE staining of tissue sections, followed by the quantitative analysis of Tb.N and Tb.Sp of HE stain ing. Scale bar = 200 μm. (H) Relative concentration of TRAP, <t>RANKL,</t> <t>CTX-1</t> and PINP in serum of each group. (I) TRAP staining of tissue sections. Scale bar = 200 μm. (J) Quantitative analysis of osteoclast number per trabecular bone surface (N. OC/BS). Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns = not statistically significant

Rankl, supplied by Elabscience Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 93 stars, based on 6 article reviews

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1) Product Images from "Ferrostatin-1 inhibits osteoclast differentiation and prevents osteoporosis by suppressing lipid peroxidation."

Article Title: Ferrostatin-1 inhibits osteoclast differentiation and prevents osteoporosis by suppressing lipid peroxidation.

Journal: Journal of orthopaedic surgery and research

doi: 10.1186/s13018-025-05544-2

Figure Legend Snippet: Fig. 7 Fer-1 inhibits osteoporosis of OVX mice. (A) Reconstruction of bone trabecular of distal femurs by Micro-CT of each group. Scale bar = 200 μm. (B-F) Quantitative micro-CT analysis of bone mineral density (BMD), bone volume/total volume ratio (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular separation (Tb.Sp). (G) HE staining of tissue sections, followed by the quantitative analysis of Tb.N and Tb.Sp of HE stain ing. Scale bar = 200 μm. (H) Relative concentration of TRAP, RANKL, CTX-1 and PINP in serum of each group. (I) TRAP staining of tissue sections. Scale bar = 200 μm. (J) Quantitative analysis of osteoclast number per trabecular bone surface (N. OC/BS). Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns = not statistically significant

Techniques Used: Micro-CT, Staining, H&E Stain, Concentration Assay

Image Search Results

Journal: Journal of orthopaedic surgery and research

Article Title: Ferrostatin-1 inhibits osteoclast differentiation and prevents osteoporosis by suppressing lipid peroxidation.

doi: 10.1186/s13018-025-05544-2

Figure Lengend Snippet: Fig. 7 Fer-1 inhibits osteoporosis of OVX mice. (A) Reconstruction of bone trabecular of distal femurs by Micro-CT of each group. Scale bar = 200 μm. (B-F) Quantitative micro-CT analysis of bone mineral density (BMD), bone volume/total volume ratio (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular separation (Tb.Sp). (G) HE staining of tissue sections, followed by the quantitative analysis of Tb.N and Tb.Sp of HE stain ing. Scale bar = 200 μm. (H) Relative concentration of TRAP, RANKL, CTX-1 and PINP in serum of each group. (I) TRAP staining of tissue sections. Scale bar = 200 μm. (J) Quantitative analysis of osteoclast number per trabecular bone surface (N. OC/BS). Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns = not statistically significant

Article Snippet: The cell supernatant or serum was collected and centrifuged for 20 min at 1000×g at 2–8°C and then added to a 96-well plate covered by ELISA antibodies against TGF-β (Elabscience, USA, E-EL-M1191), SDF-1 (Elabscience, USA, E-EL-M3046), S1P (Meimian, China, MM44778M2), PDGF-BB (Elabscience, USA, E-EL-M0632), Trap-5b (Elabscience, USA, E-EL-M1116), RANKL (Elabscience, USA, E-EL-M0644), CTX-1 (Elabscience, USA, E-EL-M3023) and PINP (Elabscience, USA, E-ELM0233).

Techniques: Micro-CT, Staining, H&E Stain, Concentration Assay

CYP11A1 catalyzes de novo steroidogenesis in osteotropic tumor cells (A and B) Representative images (A) and quantification (B) of TRAP-stained cell cultures (top) and resorption on artificial hydroxyapatite surface (bottom) by wild-type (WT) mouse bone-marrow-derived macrophages cultured for 3 days in the presence of 20 ng/mL M-CSF with or without 20 ng/mL RANKL or in the supernatants of E0771/Pa or E0771/bone cells. Scale bars represent 100 μm. (C) Heatmaps of significantly up- (red) and downregulated (blue) genes in E0771/bone cells compared to E0771/Pa cells identified by RNA sequencing, followed by moderated t test and Benjamini-Hochberg false discovery rate. (D) List of group of genes upregulated in E0771/bone cells compared to E0771/Pa cells identified by Gene Ontology and pathway enrichment analysis. (E) Analysis of gene expression of Cyp11a1 in E0771/Pa vs. E0771/bone cells. (F) Schematic representation of the role of CYP11A1 within the mitochondria of the cells. Image was created in BioRender ( www.biorender.com ). (G) Levels of pregnenolone in the supernatants of E0771/Pa, E0771/bone, and B16F10 cells measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Bar graphs show mean and SD of data from 3 independent experiments.

Journal: Cell Reports

Article Title: De novo steroidogenesis in tumor cells drives bone metastasis and osteoclastogenesis

doi: 10.1016/j.celrep.2024.113936

Figure Lengend Snippet: CYP11A1 catalyzes de novo steroidogenesis in osteotropic tumor cells (A and B) Representative images (A) and quantification (B) of TRAP-stained cell cultures (top) and resorption on artificial hydroxyapatite surface (bottom) by wild-type (WT) mouse bone-marrow-derived macrophages cultured for 3 days in the presence of 20 ng/mL M-CSF with or without 20 ng/mL RANKL or in the supernatants of E0771/Pa or E0771/bone cells. Scale bars represent 100 μm. (C) Heatmaps of significantly up- (red) and downregulated (blue) genes in E0771/bone cells compared to E0771/Pa cells identified by RNA sequencing, followed by moderated t test and Benjamini-Hochberg false discovery rate. (D) List of group of genes upregulated in E0771/bone cells compared to E0771/Pa cells identified by Gene Ontology and pathway enrichment analysis. (E) Analysis of gene expression of Cyp11a1 in E0771/Pa vs. E0771/bone cells. (F) Schematic representation of the role of CYP11A1 within the mitochondria of the cells. Image was created in BioRender ( www.biorender.com ). (G) Levels of pregnenolone in the supernatants of E0771/Pa, E0771/bone, and B16F10 cells measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Bar graphs show mean and SD of data from 3 independent experiments.

Article Snippet: Mouse Competitive ELISA Kit RANKL , Elabscience Biotech. , Cat# E-EL-M0644.

Techniques: Staining, Derivative Assay, Cell Culture, RNA Sequencing, Gene Expression, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy

Pregnenolone promotes OC development and function in vitro (A and B) Representative images (A) and quantification (B) of TRAP-stained cell cultures of WT mouse bone-marrow-derived macrophages cultured in the presence of 20 ng/mL M-CSF and 20 ng/mL RANKL or in the supernatants of E0771/bone, B16F10 WT, or Cyp11a1 −/− cells. Scale bars represent 100 μm. (C–F) Representative images (C) and quantification (D–F) of TRAP-stained cell cultures (C and D), in vitro resorption on bovine bone slices (C and E), and actin ring formation (C and F) by WT mouse bone-marrow-derived macrophages cultured for 2 days in the presence of 20 ng/mL M-CSF and 20 ng/mL RANKL and then for 3 (C, D, and F) or 11 (C and E) days in the presence of vehicle or the indicated concentration of pregnenolone. OCs are defined as TRAP-positive cells with 3 or more nuclei. Scale bars represent 100 (TRAP staining and actin ring formation) and 50 μm (bone resorption). (G) Quantification of the percentage of surviving cells detected after the binding of Annexin-V-PE (apoptosis) and 7-AAD (necrosis) markers to WT bone-marrow-derived macrophages cultured for 2 days in the presence of 20 ng/mL M-CSF and 20 ng/mL RANKL and then for another 3 days with vehicle or the indicated concentration of pregnenolone. Surviving cells are defined as negative for both Annexin-V-PE and 7-AAD staining. Bar graphs represent mean and SD of data from 3–6 independent experiments. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.002, and ∗∗∗∗ p < 0.0004; n.s., not significant.

Journal: Cell Reports

Article Title: De novo steroidogenesis in tumor cells drives bone metastasis and osteoclastogenesis

doi: 10.1016/j.celrep.2024.113936

Figure Lengend Snippet: Pregnenolone promotes OC development and function in vitro (A and B) Representative images (A) and quantification (B) of TRAP-stained cell cultures of WT mouse bone-marrow-derived macrophages cultured in the presence of 20 ng/mL M-CSF and 20 ng/mL RANKL or in the supernatants of E0771/bone, B16F10 WT, or Cyp11a1 −/− cells. Scale bars represent 100 μm. (C–F) Representative images (C) and quantification (D–F) of TRAP-stained cell cultures (C and D), in vitro resorption on bovine bone slices (C and E), and actin ring formation (C and F) by WT mouse bone-marrow-derived macrophages cultured for 2 days in the presence of 20 ng/mL M-CSF and 20 ng/mL RANKL and then for 3 (C, D, and F) or 11 (C and E) days in the presence of vehicle or the indicated concentration of pregnenolone. OCs are defined as TRAP-positive cells with 3 or more nuclei. Scale bars represent 100 (TRAP staining and actin ring formation) and 50 μm (bone resorption). (G) Quantification of the percentage of surviving cells detected after the binding of Annexin-V-PE (apoptosis) and 7-AAD (necrosis) markers to WT bone-marrow-derived macrophages cultured for 2 days in the presence of 20 ng/mL M-CSF and 20 ng/mL RANKL and then for another 3 days with vehicle or the indicated concentration of pregnenolone. Surviving cells are defined as negative for both Annexin-V-PE and 7-AAD staining. Bar graphs represent mean and SD of data from 3–6 independent experiments. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.002, and ∗∗∗∗ p < 0.0004; n.s., not significant.

Article Snippet: Mouse Competitive ELISA Kit RANKL , Elabscience Biotech. , Cat# E-EL-M0644.

Techniques: In Vitro, Staining, Derivative Assay, Cell Culture, Concentration Assay, Binding Assay

Pregnenolone drives the fusion of OCs, but it is dispensable for OC-specific gene expression (A) Gene expression in WT mouse bone-marrow-derived progenitors cultured for 2 days in the presence of 20 and 20 ng/mL M-CSF and RANKL (OCs) or M-CSF (macrophages) or pregnenolone alone and then in the indicated concentrations of pregnenolone or vehicle for 3 days. The expressions of the Nfatc1 , Acp5 , Calcr , Itgb3 , Tm7sf4 , and Ctsk genes (encoding for NFATc1, TRAP, calcitonin receptor, integrin β 3 , DC-STAMP, and cathepsin K, respectively) were determined by RT-qPCR. (B and C) Representative images (B) and quantification of the number of GFP + cells (C) generated by co-culturing bone marrow cells from Ctsk-Cre and mTmG transgenic mice in the presence of 20 or 50 ng/mL M-CSF and 20 or 50 ng/mL RANKL for 2 days and then in the presence of vehicle or 200 nM pregnenolone for another 3 days. Scale bars represent 100 μm. (D) Analysis of the number of nuclei in vehicle- or 200 nM pregnenolone-treated mouse bone-marrow-derived OC cultures. (E) Representative real-time images of Ctsk-Cre and mTmG bone marrow cells co-cultured in the presence of 20 ng/mL M-CSF and 20 ng/mL RANKL for 2 days and then in the presence of vehicle or 200 nM pregnenolone for the indicated time. Red color represents mononuclear cells; green fluorescence indicates fused OCs. Scale bars represent 20 μm. Data are from 3–6 independent experiments, with error bars representing SD. ∗ p < 0.05 and ∗∗ p < 0.01; n.s., not significant.

Journal: Cell Reports

Article Title: De novo steroidogenesis in tumor cells drives bone metastasis and osteoclastogenesis

doi: 10.1016/j.celrep.2024.113936

Figure Lengend Snippet: Pregnenolone drives the fusion of OCs, but it is dispensable for OC-specific gene expression (A) Gene expression in WT mouse bone-marrow-derived progenitors cultured for 2 days in the presence of 20 and 20 ng/mL M-CSF and RANKL (OCs) or M-CSF (macrophages) or pregnenolone alone and then in the indicated concentrations of pregnenolone or vehicle for 3 days. The expressions of the Nfatc1 , Acp5 , Calcr , Itgb3 , Tm7sf4 , and Ctsk genes (encoding for NFATc1, TRAP, calcitonin receptor, integrin β 3 , DC-STAMP, and cathepsin K, respectively) were determined by RT-qPCR. (B and C) Representative images (B) and quantification of the number of GFP + cells (C) generated by co-culturing bone marrow cells from Ctsk-Cre and mTmG transgenic mice in the presence of 20 or 50 ng/mL M-CSF and 20 or 50 ng/mL RANKL for 2 days and then in the presence of vehicle or 200 nM pregnenolone for another 3 days. Scale bars represent 100 μm. (D) Analysis of the number of nuclei in vehicle- or 200 nM pregnenolone-treated mouse bone-marrow-derived OC cultures. (E) Representative real-time images of Ctsk-Cre and mTmG bone marrow cells co-cultured in the presence of 20 ng/mL M-CSF and 20 ng/mL RANKL for 2 days and then in the presence of vehicle or 200 nM pregnenolone for the indicated time. Red color represents mononuclear cells; green fluorescence indicates fused OCs. Scale bars represent 20 μm. Data are from 3–6 independent experiments, with error bars representing SD. ∗ p < 0.05 and ∗∗ p < 0.01; n.s., not significant.

Article Snippet: Mouse Competitive ELISA Kit RANKL , Elabscience Biotech. , Cat# E-EL-M0644.

Techniques: Gene Expression, Derivative Assay, Cell Culture, Quantitative RT-PCR, Generated, Transgenic Assay, Fluorescence

Pregnenolone promotes the migration and fusion of OC precursors via P4HB (A and B) Representative images (A) and quantification (B) of wound closure by WT mouse bone-marrow-derived progenitors cultured in the presence of 20 ng/mL M-CSF and 20 ng/mL RANKL with or without 200 nM pregnenolone and with or without 3 μM quercetin-3-rutinoside for 24 h. Scale bars represent 75 μm. (C) Quantification of Transwell migration by pre-OCs cultured in the presence of 20 ng/mL M-CSF and 20 ng/mL RANKL with or without 200 nM pregnenolone and with or without 3 μM quercetin-3-rutinoside. (D) Representative images and quantification of TRAP-stained cell cultures of WT mouse bone-marrow-derived macrophages cultured in the presence of 200 nM pregnenolone with control (mock, left) or P4HB short hairpin RNA (shRNA) lentiviral particles (right). Scale bars represent 100 μm. (E) Gene expression of Tm7sf4 and Ocstamp genes (encoding for DC-STAMP and OC-STAMP, respectively) in WT mouse bone-marrow-derived progenitors cultured for 2 days in the presence of 20 and 20 ng/mL M-CSF and RANKL with or without the indicated concentration of pregnenolone for 3 days. (F) Representative histograms of the binding of α-OC-STAMP antibody to WT bone marrow cells cultured for 2 days in the presence of 20 ng/mL M-CSF and RANKL with or without 200 nM pregnenolone and/or 3 μM quercetin-3-rutinoside. Bar graphs show mean and SD of data from 3 experiments. ∗ p < 0.05; n.s., not significant.

Journal: Cell Reports

Article Title: De novo steroidogenesis in tumor cells drives bone metastasis and osteoclastogenesis

doi: 10.1016/j.celrep.2024.113936

Figure Lengend Snippet: Pregnenolone promotes the migration and fusion of OC precursors via P4HB (A and B) Representative images (A) and quantification (B) of wound closure by WT mouse bone-marrow-derived progenitors cultured in the presence of 20 ng/mL M-CSF and 20 ng/mL RANKL with or without 200 nM pregnenolone and with or without 3 μM quercetin-3-rutinoside for 24 h. Scale bars represent 75 μm. (C) Quantification of Transwell migration by pre-OCs cultured in the presence of 20 ng/mL M-CSF and 20 ng/mL RANKL with or without 200 nM pregnenolone and with or without 3 μM quercetin-3-rutinoside. (D) Representative images and quantification of TRAP-stained cell cultures of WT mouse bone-marrow-derived macrophages cultured in the presence of 200 nM pregnenolone with control (mock, left) or P4HB short hairpin RNA (shRNA) lentiviral particles (right). Scale bars represent 100 μm. (E) Gene expression of Tm7sf4 and Ocstamp genes (encoding for DC-STAMP and OC-STAMP, respectively) in WT mouse bone-marrow-derived progenitors cultured for 2 days in the presence of 20 and 20 ng/mL M-CSF and RANKL with or without the indicated concentration of pregnenolone for 3 days. (F) Representative histograms of the binding of α-OC-STAMP antibody to WT bone marrow cells cultured for 2 days in the presence of 20 ng/mL M-CSF and RANKL with or without 200 nM pregnenolone and/or 3 μM quercetin-3-rutinoside. Bar graphs show mean and SD of data from 3 experiments. ∗ p < 0.05; n.s., not significant.

Article Snippet: Mouse Competitive ELISA Kit RANKL , Elabscience Biotech. , Cat# E-EL-M0644.

Techniques: Migration, Derivative Assay, Cell Culture, Staining, Control, shRNA, Gene Expression, Concentration Assay, Binding Assay

Pregnenolone promotes human osteoclastogenesis and is secreted by human tumor cells (A–C) Representative images (A) and quantification (B and C) of TRAP-stained cell cultures of human blood mononuclear cell-derived OCs cultured for 2 days in the presence of 20 ng/mL recombinant human M-CSF and 20 ng/mL soluble human RANKL and then in the presence of the indicated concentration of pregnenolone or vehicle for 12 days. Scale bars represent 100 μm. (D) Levels of pregnenolone in the supernatants of A375, MDA-MB-435S, A549, NCI-H1299, NCI-H460, MCF-7, MDA-MB-231, DU145, LNCaP, PC-3, Hs895.T, SK-MEL-28, BEAS-2B, NCI-H128, NCI-H2126, HCC70, MCF-10A, MDA-PCa-2b, RWPE-1, and LASCPC-01 cells measured by ELISA. (E) Kaplan-Meier curve of breast-cancer-specific survival for 2,976 patients with high or low expression of Cyp11a1 in the primary tumor. Bar graphs show mean and SD of data from 3 independent experiments. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.002; n.s., not significant.

Journal: Cell Reports

Article Title: De novo steroidogenesis in tumor cells drives bone metastasis and osteoclastogenesis

doi: 10.1016/j.celrep.2024.113936

Figure Lengend Snippet: Pregnenolone promotes human osteoclastogenesis and is secreted by human tumor cells (A–C) Representative images (A) and quantification (B and C) of TRAP-stained cell cultures of human blood mononuclear cell-derived OCs cultured for 2 days in the presence of 20 ng/mL recombinant human M-CSF and 20 ng/mL soluble human RANKL and then in the presence of the indicated concentration of pregnenolone or vehicle for 12 days. Scale bars represent 100 μm. (D) Levels of pregnenolone in the supernatants of A375, MDA-MB-435S, A549, NCI-H1299, NCI-H460, MCF-7, MDA-MB-231, DU145, LNCaP, PC-3, Hs895.T, SK-MEL-28, BEAS-2B, NCI-H128, NCI-H2126, HCC70, MCF-10A, MDA-PCa-2b, RWPE-1, and LASCPC-01 cells measured by ELISA. (E) Kaplan-Meier curve of breast-cancer-specific survival for 2,976 patients with high or low expression of Cyp11a1 in the primary tumor. Bar graphs show mean and SD of data from 3 independent experiments. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.002; n.s., not significant.

Article Snippet: Mouse Competitive ELISA Kit RANKL , Elabscience Biotech. , Cat# E-EL-M0644.

Techniques: Staining, Derivative Assay, Cell Culture, Recombinant, Concentration Assay, Enzyme-linked Immunosorbent Assay, Expressing

Journal: Cell Reports

Article Title: De novo steroidogenesis in tumor cells drives bone metastasis and osteoclastogenesis

doi: 10.1016/j.celrep.2024.113936

Figure Lengend Snippet:

Article Snippet: Mouse Competitive ELISA Kit RANKL , Elabscience Biotech. , Cat# E-EL-M0644.

Techniques: Virus, shRNA, Recombinant, Modification, Staining, RNA Library Preparation, TaqMan Assay, Competitive ELISA, RNA Sequencing, Plasmid Preparation, Software, Real-time Polymerase Chain Reaction

Radiation induces bone loss and increased CR6-interacting factor-1 expression in mice. A: Micro-computed tomography images of the distal metaphysis of the femur. Mice (n = 6/group ) were exposed to Co-60 gamma rays, and received 5 Gy of whole-body sublethal irradiation at a rate of 0.69 Gy/min; B-H: Micro-computed tomography analysis of the trabecular bone volume/total volume (B), connectivity density (C), trabecular number (D), bone mineral density (E), trabecular thickness (F), trabecular spacing (G), and structure model index (H); I: Hematoxylin-eosin staining of femoral sections from irradiated mice and controls. Femoral sections from irradiated mice showed significantly decreased trabecular bone compared to controls; J: Hematoxylin-eosin staining of femoral sections from irradiated mice and controls. Femoral sections from irradiated mice showed that adipocytes increased significantly in irradiated mice; K: Tartrate-resistant acid phosphatase staining of femoral sections from irradiated mice and controls; L: Real-time quantitative polymerase chain reaction (RT-qPCR) analysis of receptor activator of nuclear factor κB ligand and osteoprotegerin mRNA expression in flushed whole bone marrow; M: Receptor activator of nuclear factor κB ligand/osteoprotegerin ratio based on RT-qPCR results; N: RT-qPCR analysis of CR6-interacting factor-1 mRNA expression in flushed whole bone marrow; O: Western blot analysis of CR6-interacting factor-1 expression in flushed whole bone marrow. aP < 0.05, bP < 0.01, and the bars represent the mean ± SD. OPG: Osteoprotegerin; RANKL: Receptor activator of nuclear factor κB ligand; Crif1: CR6-interacting factor-1; BM: Bone marrow; SMI: Structure model index; BV/TV: Bone volume/total volume; Conn.D: Connectivity density; Tb.N: Trabecular number; vBMD: Bone mineral density; Tb.Sp: Trabecular spacing; Tb.Th: Trabecular thickness.

Journal: World Journal of Stem Cells

Article Title: CR6-interacting factor-1 contributes to osteoclastogenesis by inducing receptor activator of nuclear factor κB ligand after radiation

doi: 10.4252/wjsc.v12.i3.222

Figure Lengend Snippet: Radiation induces bone loss and increased CR6-interacting factor-1 expression in mice. A: Micro-computed tomography images of the distal metaphysis of the femur. Mice (n = 6/group ) were exposed to Co-60 gamma rays, and received 5 Gy of whole-body sublethal irradiation at a rate of 0.69 Gy/min; B-H: Micro-computed tomography analysis of the trabecular bone volume/total volume (B), connectivity density (C), trabecular number (D), bone mineral density (E), trabecular thickness (F), trabecular spacing (G), and structure model index (H); I: Hematoxylin-eosin staining of femoral sections from irradiated mice and controls. Femoral sections from irradiated mice showed significantly decreased trabecular bone compared to controls; J: Hematoxylin-eosin staining of femoral sections from irradiated mice and controls. Femoral sections from irradiated mice showed that adipocytes increased significantly in irradiated mice; K: Tartrate-resistant acid phosphatase staining of femoral sections from irradiated mice and controls; L: Real-time quantitative polymerase chain reaction (RT-qPCR) analysis of receptor activator of nuclear factor κB ligand and osteoprotegerin mRNA expression in flushed whole bone marrow; M: Receptor activator of nuclear factor κB ligand/osteoprotegerin ratio based on RT-qPCR results; N: RT-qPCR analysis of CR6-interacting factor-1 mRNA expression in flushed whole bone marrow; O: Western blot analysis of CR6-interacting factor-1 expression in flushed whole bone marrow. aP < 0.05, bP < 0.01, and the bars represent the mean ± SD. OPG: Osteoprotegerin; RANKL: Receptor activator of nuclear factor κB ligand; Crif1: CR6-interacting factor-1; BM: Bone marrow; SMI: Structure model index; BV/TV: Bone volume/total volume; Conn.D: Connectivity density; Tb.N: Trabecular number; vBMD: Bone mineral density; Tb.Sp: Trabecular spacing; Tb.Th: Trabecular thickness.

Article Snippet: The concentrations of RANKL and OPG were measured using the Mouse RANKL ELISA Kit (E-EL-M0644c, elabscience), Human Soluble Receptor Activator of Nuclear Factor-kB Ligand ELISA Kit (E-EL-H5558c, elabscience), Mouse OPG ELISA Kit (E-EL-M0081c, elabscience), and Human OPG ELISA Kit (E-EL-H1341c, elabscience) according to the manufacturer’s instructions.

Techniques: Expressing, Micro-CT, Irradiation, Staining, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot

Overexpression of CR6-interacting factor-1 in bone marrow mesenchymal stem/stromal cells increases receptor activator of nuclear factor κB ligand secretion and osteoclastogenesis. A: Western blot analysis of CR6-interacting factor-1 (Crif1) expression in mouse bone marrow mesenchymal stem/stromal cells (BM-MSCs). Mouse BM-MSCs were transfected with a Crif1 lentiviral overexpression vector; B: Real-time quantitative polymerase chain reaction analysis of receptor activator of nuclear factor κB ligand (RANKL) and osteoprotegerin (OPG) mRNA expression in BM-MSCs and Crif1-overexpressing BM-MSCs. BM-MSCs and Crif1-overexpressing BM-MSCs were cocultured with RAW264.7, respectively; C: RANKL/OPG ratio based on real-time quantitative polymerase chain reaction results; D: Enzyme linked immunosorbent assay analysis of RANKL protein levels in coculture supernatant medium; E: Enzyme linked immunosorbent assay analysis of OPG protein levels in coculture supernatant medium; F: RANKL/OPG ratio in coculture supernatant medium; G: Tartrate-resistant acid phosphatase staining of RAW264.7 cells after 7 d of coculture; H: Average number of tartrate-resistant acid phosphatase-positive cells/well (arrow) from RAW264.7 cells in coculture. aP < 0.05, bP < 0.01, and the bars represent the mean ± SD. OPG: Osteoprotegerin; RANKL: Receptor activator of nuclear factor κB ligand; Crif1: CR6-interacting factor-1; BM-MSCs: Bone marrow mesenchymal stem/stromal cells; BM-MSCs-OV: Crif1-overexpressing BM-MSCs.

Journal: World Journal of Stem Cells

Article Title: CR6-interacting factor-1 contributes to osteoclastogenesis by inducing receptor activator of nuclear factor κB ligand after radiation

doi: 10.4252/wjsc.v12.i3.222

Figure Lengend Snippet: Overexpression of CR6-interacting factor-1 in bone marrow mesenchymal stem/stromal cells increases receptor activator of nuclear factor κB ligand secretion and osteoclastogenesis. A: Western blot analysis of CR6-interacting factor-1 (Crif1) expression in mouse bone marrow mesenchymal stem/stromal cells (BM-MSCs). Mouse BM-MSCs were transfected with a Crif1 lentiviral overexpression vector; B: Real-time quantitative polymerase chain reaction analysis of receptor activator of nuclear factor κB ligand (RANKL) and osteoprotegerin (OPG) mRNA expression in BM-MSCs and Crif1-overexpressing BM-MSCs. BM-MSCs and Crif1-overexpressing BM-MSCs were cocultured with RAW264.7, respectively; C: RANKL/OPG ratio based on real-time quantitative polymerase chain reaction results; D: Enzyme linked immunosorbent assay analysis of RANKL protein levels in coculture supernatant medium; E: Enzyme linked immunosorbent assay analysis of OPG protein levels in coculture supernatant medium; F: RANKL/OPG ratio in coculture supernatant medium; G: Tartrate-resistant acid phosphatase staining of RAW264.7 cells after 7 d of coculture; H: Average number of tartrate-resistant acid phosphatase-positive cells/well (arrow) from RAW264.7 cells in coculture. aP < 0.05, bP < 0.01, and the bars represent the mean ± SD. OPG: Osteoprotegerin; RANKL: Receptor activator of nuclear factor κB ligand; Crif1: CR6-interacting factor-1; BM-MSCs: Bone marrow mesenchymal stem/stromal cells; BM-MSCs-OV: Crif1-overexpressing BM-MSCs.

Techniques: Over Expression, Western Blot, Expressing, Transfection, Plasmid Preparation, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Staining

CR6-interacting factor-1 mediates adipogenesis and receptor activator of nuclear factor κB ligand secretion in adipocytes. A: Oil red O staining analysis of mouse bone marrow mesenchymal stem/stromal cells (BM-MSCs) after 21 d of adipogenic differentiation. Crif1 was knocked out in mouse BM-MSCs (BM-MSCs-KO), and knockout cells and controls were irradiated with 9 Gy of Co-60, and then treated with mouse mesenchymal stem cell adipogenic differentiation medium (Ad) to induce adipogenesis; B: The dye from oil red O staining was extracted using isopropanol, and the optical density at 510 nm was measured using Benchmark Plus; C: Western blot analysis of adipogenesis-related markers and transcription factors PPARγ and AP2 in mouse BM-MSCs after 21 d of adipogenic differentiation; D: Real-time quantitative polymerase chain reaction analysis of receptor activator of nuclear factor κB ligand (RANKL) and osteoprotegerin (OPG) mRNA expression in BM-MSCs and BM-MSCs-KO; E: RANKL/OPG ratio based on real-time quantitative polymerase chain reaction results; F: Enzyme linked immunosorbent assay analysis of RANKL protein levels in supernatant Ad; G: Enzyme linked immunosorbent assay analysis of OPG protein levels in supernatant Ad; H: RANKL/OPG ratio in supernatant Ad. aP < 0.05 vs control (BM-MSCs), bP < 0.01 vs control (BM-MSCs); dP < 0.01 between 9 Gy-BM-MSCs and 9 Gy-BM-MSCs-KO, and the bars represent the mean ± SD. BM-MSCs: Bone marrow mesenchymal stem/stromal cells; BM-MSCs-KO: Crif1 knockout mouse BM-MSCs; OPG: Osteoprotegerin; RANKL: Receptor activator of nuclear factor κB ligand.

Journal: World Journal of Stem Cells

Article Title: CR6-interacting factor-1 contributes to osteoclastogenesis by inducing receptor activator of nuclear factor κB ligand after radiation

doi: 10.4252/wjsc.v12.i3.222

Figure Lengend Snippet: CR6-interacting factor-1 mediates adipogenesis and receptor activator of nuclear factor κB ligand secretion in adipocytes. A: Oil red O staining analysis of mouse bone marrow mesenchymal stem/stromal cells (BM-MSCs) after 21 d of adipogenic differentiation. Crif1 was knocked out in mouse BM-MSCs (BM-MSCs-KO), and knockout cells and controls were irradiated with 9 Gy of Co-60, and then treated with mouse mesenchymal stem cell adipogenic differentiation medium (Ad) to induce adipogenesis; B: The dye from oil red O staining was extracted using isopropanol, and the optical density at 510 nm was measured using Benchmark Plus; C: Western blot analysis of adipogenesis-related markers and transcription factors PPARγ and AP2 in mouse BM-MSCs after 21 d of adipogenic differentiation; D: Real-time quantitative polymerase chain reaction analysis of receptor activator of nuclear factor κB ligand (RANKL) and osteoprotegerin (OPG) mRNA expression in BM-MSCs and BM-MSCs-KO; E: RANKL/OPG ratio based on real-time quantitative polymerase chain reaction results; F: Enzyme linked immunosorbent assay analysis of RANKL protein levels in supernatant Ad; G: Enzyme linked immunosorbent assay analysis of OPG protein levels in supernatant Ad; H: RANKL/OPG ratio in supernatant Ad. aP < 0.05 vs control (BM-MSCs), bP < 0.01 vs control (BM-MSCs); dP < 0.01 between 9 Gy-BM-MSCs and 9 Gy-BM-MSCs-KO, and the bars represent the mean ± SD. BM-MSCs: Bone marrow mesenchymal stem/stromal cells; BM-MSCs-KO: Crif1 knockout mouse BM-MSCs; OPG: Osteoprotegerin; RANKL: Receptor activator of nuclear factor κB ligand.

Techniques: Staining, Knock-Out, Irradiation, Western Blot, Real-time Polymerase Chain Reaction, Expressing, Enzyme-linked Immunosorbent Assay, Control

CR6-interacting factor-1 is involved in the regulation of receptor activator of nuclear factor κB ligand expression after radiation. A: Western blot analysis of CR6-interacting factor-1 (Crif1) and receptor activator of nuclear factor κB expression in RAW264.7 cells. Crif1 was knocked out in RAW264.7 cells (RAW264.7-KO); B: Tartrate-resistant acid phosphatase (TRAP) staining of RAW264.7-KO and controls after 7 d of coculture with mouse bone marrow mesenchymal stem/stromal cells (BM-MSCs); C: Average number of TRAP-positive cells/well (arrow) from RAW264.7-KO and controls after 7 d of coculture with mouse BM-MSCs; D: Western blot analysis of Crif1 expression in BM-MSCs. Crif1 was knocked out in mouse BM-MSCs (BM-MSCs-KO), and BM-MSCs-KO and controls were irradiated with Co-60 at a single dose of 9 Gy; E: Real-time quantitative polymerase chain reaction analysis of receptor activator of nuclear factor κB ligand (RANKL) and osteoprotegerin (OPG) mRNA expression in BM-MSCs and BM-MSCs-KO. BM-MSCs and BM-MSCs-KO were cocultured with RAW264.7; F: RANKL/OPG ratio based on real-time quantitative polymerase chain reaction results; G: Enzyme linked immunosorbent assay analysis of RANKL protein levels in coculture supernatant medium; H: Enzyme linked immunosorbent assay analysis of OPG protein levels in coculture supernatant medium; I: RANKL/OPG ratio in coculture supernatant medium; J: TRAP staining of RAW264.7 after 7 d of coculture; K: Average number of TRAP-positive cells/well (arrow) from RAW264.7 in coculture. aP < 0.05 vs control (BM-MSCs), bP < 0.01 vs control (BM-MSCs); dP < 0.01 between 9 Gy-BM-MSCs and 9 Gy-BM-MSCs-KO, and the bars represent the mean ± standard deviation. BM-MSCs: Bone marrow mesenchymal stem/stromal cells; BM-MSCs-KO: Crif1 was knocked out from mouse BM-MSCs; RAW264.7-KO: Crif1 was knocked out from RAW264.7 cells; OPG: Osteoprotegerin; RANKL: Receptor activator of nuclear factor κB ligand.

Journal: World Journal of Stem Cells

Article Title: CR6-interacting factor-1 contributes to osteoclastogenesis by inducing receptor activator of nuclear factor κB ligand after radiation

doi: 10.4252/wjsc.v12.i3.222

Figure Lengend Snippet: CR6-interacting factor-1 is involved in the regulation of receptor activator of nuclear factor κB ligand expression after radiation. A: Western blot analysis of CR6-interacting factor-1 (Crif1) and receptor activator of nuclear factor κB expression in RAW264.7 cells. Crif1 was knocked out in RAW264.7 cells (RAW264.7-KO); B: Tartrate-resistant acid phosphatase (TRAP) staining of RAW264.7-KO and controls after 7 d of coculture with mouse bone marrow mesenchymal stem/stromal cells (BM-MSCs); C: Average number of TRAP-positive cells/well (arrow) from RAW264.7-KO and controls after 7 d of coculture with mouse BM-MSCs; D: Western blot analysis of Crif1 expression in BM-MSCs. Crif1 was knocked out in mouse BM-MSCs (BM-MSCs-KO), and BM-MSCs-KO and controls were irradiated with Co-60 at a single dose of 9 Gy; E: Real-time quantitative polymerase chain reaction analysis of receptor activator of nuclear factor κB ligand (RANKL) and osteoprotegerin (OPG) mRNA expression in BM-MSCs and BM-MSCs-KO. BM-MSCs and BM-MSCs-KO were cocultured with RAW264.7; F: RANKL/OPG ratio based on real-time quantitative polymerase chain reaction results; G: Enzyme linked immunosorbent assay analysis of RANKL protein levels in coculture supernatant medium; H: Enzyme linked immunosorbent assay analysis of OPG protein levels in coculture supernatant medium; I: RANKL/OPG ratio in coculture supernatant medium; J: TRAP staining of RAW264.7 after 7 d of coculture; K: Average number of TRAP-positive cells/well (arrow) from RAW264.7 in coculture. aP < 0.05 vs control (BM-MSCs), bP < 0.01 vs control (BM-MSCs); dP < 0.01 between 9 Gy-BM-MSCs and 9 Gy-BM-MSCs-KO, and the bars represent the mean ± standard deviation. BM-MSCs: Bone marrow mesenchymal stem/stromal cells; BM-MSCs-KO: Crif1 was knocked out from mouse BM-MSCs; RAW264.7-KO: Crif1 was knocked out from RAW264.7 cells; OPG: Osteoprotegerin; RANKL: Receptor activator of nuclear factor κB ligand.

Techniques: Expressing, Western Blot, Staining, Irradiation, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Control, Standard Deviation

CR6-interacting factor-1 promotes receptor activator of nuclear factor κB ligand secretion by modulating the cAMP/PKA signaling pathway. A: Real-time quantitative polymerase chain reaction (RT-qPCR) analysis of receptor activator of nuclear factor κB ligand (RANKL) and osteoprotegerin (OPG) mRNA expression in bone marrow mesenchymal stem/stromal cells (BM-MSCs) and Crif1 knockout BM-MSCs treated with 25 µmol/L forskolin in the coculture with RAW264.7; B: RANKL/OPG ratio based on RT-qPCR results; C: Enzyme linked immunosorbent assay (ELISA) analysis of RANKL levels in coculture supernatant medium treated with 25 µmol/L forskolin; D: ELISA analysis of OPG levels in coculture supernatant medium treated with 25 µmol/L forskolin; E: RANKL/OPG ratio in coculture supernatant medium treated with 25 µmol/L forskolin; F: RT-qPCR analysis of RANKL and OPG mRNA expression in BM-MSCs and BM-MSCs-OV treated with 20 µmol/L H-89 in the coculture with RAW264.7; G: RANKL/OPG ratio based on RT-qPCR results; H: ELISA analysis of RANKL levels in coculture supernatant medium treated with 20 µmol/L H-89; I: ELISA analysis of OPG levels in coculture supernatant medium treated with 20 µmol/L H-89; J: RANKL/OPG ratio in coculture supernatant medium treated with 20 µmol/L H-89; K: Tartrate-resistant acid phosphatase (TRAP) staining of RAW264.7 cells in coculture treated with 25 µmol/L forskolin; L: Average number of TRAP-positive cells/well (arrow) from RAW264.7 cells in coculture treated with 25 µmol/L forskolin; M: Western blot analysis of phospho-cyclic adenosine monophosphate response element-binding protein phosphorylation levels in BM-MSCs in coculture treated with 25 µmol/L forskolin; N: TRAP staining of RAW264.7 in coculture treated with 20 µmol/L H-89; O: Average number of TRAP-positive cells/well (arrow) from RAW264.7 in coculture treated with 20 µmol/L H-89; P: Western blot analysis of phospho-cyclic adenosine monophosphate response element-binding protein phosphorylation levels in BM-MSCs in coculture treated with 20 µmol/L H-89. bP < 0.01 vs control (BM-MSCs); dP < 0.01 between between BM-MSCs treated with 25 µmol/L forskolin and Crif1 knockout BM-MSCs treated with 25 µmol/L forskolin; fP < 0.01 between Crif1-overexpressing BM-MSCs and Crif1-overexpressing BM-MSCs treated with 20 µmol/L H-89, and the bars represent the mean ± SD. BM-MSCs: Bone marrow mesenchymal stem/stromal cells; BM-MSCs-KO: Crif1 knockout BM-MSCs; OPG: Osteoprotegerin; RANKL: Receptor activator of nuclear factor κB ligand; CREB: Cyclic adenosine monophosphate response element-binding protein.

Journal: World Journal of Stem Cells

Article Title: CR6-interacting factor-1 contributes to osteoclastogenesis by inducing receptor activator of nuclear factor κB ligand after radiation

doi: 10.4252/wjsc.v12.i3.222

Figure Lengend Snippet: CR6-interacting factor-1 promotes receptor activator of nuclear factor κB ligand secretion by modulating the cAMP/PKA signaling pathway. A: Real-time quantitative polymerase chain reaction (RT-qPCR) analysis of receptor activator of nuclear factor κB ligand (RANKL) and osteoprotegerin (OPG) mRNA expression in bone marrow mesenchymal stem/stromal cells (BM-MSCs) and Crif1 knockout BM-MSCs treated with 25 µmol/L forskolin in the coculture with RAW264.7; B: RANKL/OPG ratio based on RT-qPCR results; C: Enzyme linked immunosorbent assay (ELISA) analysis of RANKL levels in coculture supernatant medium treated with 25 µmol/L forskolin; D: ELISA analysis of OPG levels in coculture supernatant medium treated with 25 µmol/L forskolin; E: RANKL/OPG ratio in coculture supernatant medium treated with 25 µmol/L forskolin; F: RT-qPCR analysis of RANKL and OPG mRNA expression in BM-MSCs and BM-MSCs-OV treated with 20 µmol/L H-89 in the coculture with RAW264.7; G: RANKL/OPG ratio based on RT-qPCR results; H: ELISA analysis of RANKL levels in coculture supernatant medium treated with 20 µmol/L H-89; I: ELISA analysis of OPG levels in coculture supernatant medium treated with 20 µmol/L H-89; J: RANKL/OPG ratio in coculture supernatant medium treated with 20 µmol/L H-89; K: Tartrate-resistant acid phosphatase (TRAP) staining of RAW264.7 cells in coculture treated with 25 µmol/L forskolin; L: Average number of TRAP-positive cells/well (arrow) from RAW264.7 cells in coculture treated with 25 µmol/L forskolin; M: Western blot analysis of phospho-cyclic adenosine monophosphate response element-binding protein phosphorylation levels in BM-MSCs in coculture treated with 25 µmol/L forskolin; N: TRAP staining of RAW264.7 in coculture treated with 20 µmol/L H-89; O: Average number of TRAP-positive cells/well (arrow) from RAW264.7 in coculture treated with 20 µmol/L H-89; P: Western blot analysis of phospho-cyclic adenosine monophosphate response element-binding protein phosphorylation levels in BM-MSCs in coculture treated with 20 µmol/L H-89. bP < 0.01 vs control (BM-MSCs); dP < 0.01 between between BM-MSCs treated with 25 µmol/L forskolin and Crif1 knockout BM-MSCs treated with 25 µmol/L forskolin; fP < 0.01 between Crif1-overexpressing BM-MSCs and Crif1-overexpressing BM-MSCs treated with 20 µmol/L H-89, and the bars represent the mean ± SD. BM-MSCs: Bone marrow mesenchymal stem/stromal cells; BM-MSCs-KO: Crif1 knockout BM-MSCs; OPG: Osteoprotegerin; RANKL: Receptor activator of nuclear factor κB ligand; CREB: Cyclic adenosine monophosphate response element-binding protein.

Techniques: Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Knock-Out, Enzyme-linked Immunosorbent Assay, Staining, Western Blot, Binding Assay, Phospho-proteomics, Control

CR6-interacting factor-1 inhibitors effectively suppress receptor activator of nuclear factor κB ligand secretion and adipogenesis. A: Enzyme linked immunosorbent assay (ELISA) analysis of receptor activator of nuclear factor κB ligand protein levels in the supernatant medium. Human bone marrow mesenchymal stem/stromal cells (H-BM-MSCs) were pretreated with five different compounds (25 µmol/L) followed by treatment with forskolin (25 µmol/L), and supernatant medium was collected for ELISA after 3 d; B: ELISA analysis of osteoprotegerin protein levels in supernatant medium; C: Receptor activator of nuclear factor κB ligand/osteoprotegerin ratio in supernatant medium; D: Oil red O staining analysis of H-BM-MSCs after 21 d of adipogenic differentiation. H-BM-MSCs were pretreated with five different compounds (25 µmol/L) followed by adipogenic induction; E: The dye from oil red O staining was extracted using isopropanol, and the optical density at 510 nm was measured using Benchmark Plus; F: Western blot analysis of cyclic adenosine monophosphate response element-binding protein phosphorylation levels. H-BM-MSCs were pretreated with five different compounds (25 µmol/L) followed by treatment with forskolin (25 µmol/L) and total protein lysates were extracted for cyclic adenosine monophosphate response element-binding protein phosphorylation detection after 1 h. aP < 0.05, bP < 0.01, and the bars represent the mean ± SD. OPG: Osteoprotegerin; RANKL: Receptor activator of nuclear factor κB ligand; CREB: Cyclic adenosine monophosphate response element-binding protein; H-BM-MSCs: Human bone marrow mesenchymal stem/stromal cells.

Journal: World Journal of Stem Cells

Article Title: CR6-interacting factor-1 contributes to osteoclastogenesis by inducing receptor activator of nuclear factor κB ligand after radiation

doi: 10.4252/wjsc.v12.i3.222

Figure Lengend Snippet: CR6-interacting factor-1 inhibitors effectively suppress receptor activator of nuclear factor κB ligand secretion and adipogenesis. A: Enzyme linked immunosorbent assay (ELISA) analysis of receptor activator of nuclear factor κB ligand protein levels in the supernatant medium. Human bone marrow mesenchymal stem/stromal cells (H-BM-MSCs) were pretreated with five different compounds (25 µmol/L) followed by treatment with forskolin (25 µmol/L), and supernatant medium was collected for ELISA after 3 d; B: ELISA analysis of osteoprotegerin protein levels in supernatant medium; C: Receptor activator of nuclear factor κB ligand/osteoprotegerin ratio in supernatant medium; D: Oil red O staining analysis of H-BM-MSCs after 21 d of adipogenic differentiation. H-BM-MSCs were pretreated with five different compounds (25 µmol/L) followed by adipogenic induction; E: The dye from oil red O staining was extracted using isopropanol, and the optical density at 510 nm was measured using Benchmark Plus; F: Western blot analysis of cyclic adenosine monophosphate response element-binding protein phosphorylation levels. H-BM-MSCs were pretreated with five different compounds (25 µmol/L) followed by treatment with forskolin (25 µmol/L) and total protein lysates were extracted for cyclic adenosine monophosphate response element-binding protein phosphorylation detection after 1 h. aP < 0.05, bP < 0.01, and the bars represent the mean ± SD. OPG: Osteoprotegerin; RANKL: Receptor activator of nuclear factor κB ligand; CREB: Cyclic adenosine monophosphate response element-binding protein; H-BM-MSCs: Human bone marrow mesenchymal stem/stromal cells.

Techniques: Enzyme-linked Immunosorbent Assay, Staining, Western Blot, Binding Assay, Phospho-proteomics

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