Journal: Biochemistry and Biophysics Reports

Article Title: TNFα stimulates osteoclastogenesis and expression of CX3CL1 in non-adherent bone marrow cells

doi: 10.1016/j.bbrep.2025.102155

Figure Lengend Snippet: TNFα induced osteoclast formation at the late culture time point in the presence of RANKL and M-CSF. (A) Cultured bone marrow cells were treated with 100 ng/mL RANKL, 20 ng/mL M-CSF, and indicated doses of TNFα (1, 3, or 10 ng/mL) for 10 days. (B) Cultured bone marrow cells were treated with 100 ng/mL RANKL and 20 ng/mL M-CSF for 10 days. TNFα (10 ng/mL) was also added in the early (days 1–4), middle (days 4–9), and late (days 7–9) stages of cell culture. Cells were assessed by TRAP staining, and TRAP-positive multinuclear cells containing more than three nuclei (TRAP[+] MNCs) were counted. n = 8 per group, ∗ p < 0.05 compared with the control. Images are shown at low ( × 100) and high ( × 400) magnification to visualize the overall cell density and detailed morphology of osteoclasts, respectively. Scale bars = 0.25 mm.

Article Snippet: Recombinant soluble human RANKL was prepared by Oriental Yeast Co., Ltd. (Tokyo, Japan).

Techniques: Cell Culture, Staining, Control

Journal: Biochemistry and Biophysics Reports

Article Title: TNFα stimulates osteoclastogenesis and expression of CX3CL1 in non-adherent bone marrow cells

doi: 10.1016/j.bbrep.2025.102155

Figure Lengend Snippet: TNFα mRNA expression level was higher in non-adherent bone marrow cells, whereas TNFR1 and TNFR2 mRNA expression levels were higher in osteoclasts. Cultured bone marrow cells were treated with 100 ng/mL RANKL and 20 ng/mL M-CSF for 9 days. Cells were separated into osteoclasts and non-adherent bone marrow cells using the pronase procedure (A). RANKL and RANK (B), and TNFα, TNFR1, and TNFR2 (C) mRNA expression levels were quantified by quantitative RT-PCR. mRNA expression levels were normalized to that of β-actin mRNA. n = 3 per group, ∗ p < 0.05 compared with osteoclasts.

Article Snippet: Recombinant soluble human RANKL was prepared by Oriental Yeast Co., Ltd. (Tokyo, Japan).

Techniques: Expressing, Cell Culture, Quantitative RT-PCR

Journal: Biochemistry and Biophysics Reports

Article Title: TNFα stimulates osteoclastogenesis and expression of CX3CL1 in non-adherent bone marrow cells

doi: 10.1016/j.bbrep.2025.102155

Figure Lengend Snippet: TNFα treatment in the later stage of culture increased CX3CL1 and CXCL7 mRNA expression levels in non-adherent bone marrow cells. Cultured bone marrow cells were treated with 100 ng/mL RANKL and 20 ng/mL M-CSF for 9 days. TNFα (10 ng/mL) was also added in the late (days 7–9) stage of cell culture. Cells were separated into osteoclasts and non-adherent bone marrow cells using the pronase procedure. RANK, CXCR4, CXCR2, IGF1R, NFATc1, DC-STAMP, TRAP, CX3CR1, and iNOS mRNA expression levels in osteoclasts (A) and RANKL, SDF1, CXCL1, CXCL7, IGF2, OPG, and iNOS mRNA expression levels in non-adherent bone marrow cells were quantified using quantitative RT-PCR. mRNA expression levels were normalized to that of β-actin mRNA. n = 3 per group, ∗ p < 0.05 compared with the control (without TNFα stimulation).

Article Snippet: Recombinant soluble human RANKL was prepared by Oriental Yeast Co., Ltd. (Tokyo, Japan).

Techniques: Expressing, Cell Culture, Quantitative RT-PCR, Control

Journal: Biochemistry and Biophysics Reports

Article Title: TNFα stimulates osteoclastogenesis and expression of CX3CL1 in non-adherent bone marrow cells

doi: 10.1016/j.bbrep.2025.102155

Figure Lengend Snippet: TNFR1-and TNFR2-neutralizing antibodies reduced the enhancement of osteoclast formation by TNFα treatment in the later stage of cell culture. (A) Cultured bone marrow cells were treated with 100 ng/mL RANKL and 20 ng/mL M-CSF for 9 days. TNFα (10 ng/mL) was also added in the late (days 7–9) stage of cell culture. Cells were also treated with anti-TNFR1 and/or anti-TNFR2 antibody (Ab) (each 3 μg/mL) 30 min before TNFα treatment. Cells were assessed by TRAP staining, and TRAP[+] MNCs were counted. n = 4 per group, ∗ p < 0.05 compared with the control (without TNFα and without anti-TNFR1 and/or anti-TNFR2 antibody stimulation). # p < 0.05 compared with TNFα alone. Images are shown at low ( × 100) and high ( × 400) magnification to visualize the overall cell density and detailed morphology of osteoclasts, respectively. Scale bars = 0.25 mm. (B) CX3CL1 and CXCL7 mRNA expression levels in non-adherent bone marrow cells were quantified using quantitative RT-PCR. mRNA expression levels were normalized to that of β-actin mRNA. n = 3 per group, ∗ p < 0.05 compared with the control (without TNFα stimulation).

Article Snippet: Recombinant soluble human RANKL was prepared by Oriental Yeast Co., Ltd. (Tokyo, Japan).

Techniques: Cell Culture, Staining, Control, Expressing, Quantitative RT-PCR

Journal: Research

Article Title: Visomitin Attenuates Pathological Bone Loss by Reprogramming Osteoclast Metabolism via the STAT3/LDHB Axis

doi: 10.34133/research.0784

Figure Lengend Snippet: Visomitin diminishes the intracellular ROS levels and attenuates osteoclastogenesis. (A) The relative antioxidant capacity of indicated antioxidants at 300 nm was evaluated in the ABTS system ( n = 3). (B and C) Evaluation and quantification of the intracellular ROS levels of BMMs exposed to H 2 O 2 after pretreatment of a range of antioxidants (300 nm) using flow cytometry ( n = 3). (D and E) Detection and quantification of the mean fluorescence intensity (MFI) of DCFH-DA probe in BMMs following treatment with either RANKL or Visomitin; scale bars, 100 μm ( n = 5). (F and G) Detection and quantification of the MFI of MitoSOX probe in BMMs following treatment with either RANKL or Visomitin; scale bars, 50 μm ( n = 5). (H) BMMs were treated with different dosages of Visomitin and subjected to in vitro osteoclast differentiation. Representative images of TRAP staining were shown. Scale bars, 50 μm. (I) Quantification of TRAP + multinuclear cells per well in panel (A) ( n = 3). (J) BMMs were subjected to in vitro osteoclast differentiation and treated with 300 nm Visomitin at specified stages. Representative images of TRAP staining were shown. Scale bars, 50 μm. (K) Quantification of TRAP + multinuclear cells per well in panel (C) ( n = 3). (L) Representative images of wheat germ agglutinin (WGA) staining in osteoclasts treated with or without Visomitin. Scale bars, 10 μm. (M) Quantification of bone pit depth in panel (I) ( n = 12). (N) Representative SEM images of bone slice resorption pits. Scale bars, 10 μm. (O) Quantification of bone resorption pit area ( n = 6). Data are mean ± SD; * P < 0.05, ** P < 0.01, and *** P < 0.001; ns, not significant.

Article Snippet: Recombinant soluble mouse macrophage colony-stimulating factor (M-CSF) (Cat. No. CB34) was supplied by Novoprotein; Recombinant soluble mouse RANKL (Cat. No. 462-TEC-010) were purchased from R&D Systems; Total Antioxidant Capacity (T-AOC) Assay Kit (Cat. No. S0121), ROS Assay Kit (Cat. No. S0033S), Mitochondrial membrane potential assay kit with JC-1 (Cat. No. C2006), MitoSOX Assay Kit (Cat. No. S0061S), DHE (Cat. No. S0063), and Antifade Mounting Medium (Cat. No. P0126) were supplied by Beyotime. iF488-wheat germ agglutinin (Cat. No. G1730) was purchased from Servicebio.

Techniques: Flow Cytometry, Fluorescence, In Vitro, Staining

Journal: Research

Article Title: Visomitin Attenuates Pathological Bone Loss by Reprogramming Osteoclast Metabolism via the STAT3/LDHB Axis

doi: 10.34133/research.0784

Figure Lengend Snippet: Visomitin attenuates the activation of RANKL-RANK signaling pathways. BMMs treated with Visomitin or PBS were subjected to osteoclast differentiation, followed by Transcriptome RNA-seq. Genes with |log 2 FC| > 1, P < 0.05, and TPM > 0.5 are designated as differentially expressed genes (DEGs) ( n = 3). (A) The heatmap illustrating the gene expression profiles derived from RNA-seq data. (B) Analysis of RNA-seq data for cell and tissue specificity utilizing the PaGenBase database. (C) The volcano plot illustrating the gene expression profiles derived from RNA-seq data. (D) KEGG enrichment analysis of DEGs obtained from RNA-seq data. (E) GO enrichment analysis of down-regulated genes (Log 2 FC < −1 and P < 0.05) obtained from RNA-seq data. (F) GSEA of Gene Ontology Biological Processes in RNA-seq data. (G) Network of enrich terms derived from RNA-seq data utilizing the Metascape database. (H) Representative immunoblots illustrating the effects of Visomitin on the activation of RANKL-RANK signaling pathways, including NF-κB, MAPK, and AKT pathways ( n = 3). Data are mean ± SD; * P < 0.05, ** P < 0.01, and *** P < 0.001; ns, not significant.

Article Snippet: Recombinant soluble mouse macrophage colony-stimulating factor (M-CSF) (Cat. No. CB34) was supplied by Novoprotein; Recombinant soluble mouse RANKL (Cat. No. 462-TEC-010) were purchased from R&D Systems; Total Antioxidant Capacity (T-AOC) Assay Kit (Cat. No. S0121), ROS Assay Kit (Cat. No. S0033S), Mitochondrial membrane potential assay kit with JC-1 (Cat. No. C2006), MitoSOX Assay Kit (Cat. No. S0061S), DHE (Cat. No. S0063), and Antifade Mounting Medium (Cat. No. P0126) were supplied by Beyotime. iF488-wheat germ agglutinin (Cat. No. G1730) was purchased from Servicebio.

Techniques: Activation Assay, Protein-Protein interactions, RNA Sequencing, Gene Expression, Derivative Assay, Western Blot

Journal: Research

Article Title: Visomitin Attenuates Pathological Bone Loss by Reprogramming Osteoclast Metabolism via the STAT3/LDHB Axis

doi: 10.34133/research.0784

Figure Lengend Snippet: Visomitin reprograms energy metabolism during osteoclastogenesis. (A) GSEA analysis of Reactome and KEGG pathways in RNA-seq data. (B) The heatmap depicting the gene expression profiles of the 2 pathways presented in panel (A). (C) Representative JC-1 staining of BMMs treated with MCSF, RANKL, or Visomitin as indicated. (D) Quantification of JC-1 staining as presented in panel (C) ( n = 5). (E) Intracellular ATP levels following treatment with MCSF, RANKL, or Visomitin as indicated ( n = 5). (F) Representative immunoblots illustrating the expression levels of proteins within the aforementioned 2 pathways following Visomitin treatment ( n = 3). (G and H) Representative Seahorse graphs for oxygen consumption rate (OCR) or extracellular acidification rate (ECAR) ( n = 3). (I) Quantification of the basal respiration, maximal respiration, and ATP production from OCR data. (J) Quantification of the non-glycolytic acidification, glycolysis, and glycolytic capacity from ECAR data. Data are mean ± SD; * P < 0.05, ** P < 0.01, and *** P < 0.001; ns, not significant.

Article Snippet: Recombinant soluble mouse macrophage colony-stimulating factor (M-CSF) (Cat. No. CB34) was supplied by Novoprotein; Recombinant soluble mouse RANKL (Cat. No. 462-TEC-010) were purchased from R&D Systems; Total Antioxidant Capacity (T-AOC) Assay Kit (Cat. No. S0121), ROS Assay Kit (Cat. No. S0033S), Mitochondrial membrane potential assay kit with JC-1 (Cat. No. C2006), MitoSOX Assay Kit (Cat. No. S0061S), DHE (Cat. No. S0063), and Antifade Mounting Medium (Cat. No. P0126) were supplied by Beyotime. iF488-wheat germ agglutinin (Cat. No. G1730) was purchased from Servicebio.

Techniques: RNA Sequencing, Gene Expression, Staining, Western Blot, Expressing

Journal: Research

Article Title: Visomitin Attenuates Pathological Bone Loss by Reprogramming Osteoclast Metabolism via the STAT3/LDHB Axis

doi: 10.34133/research.0784

Figure Lengend Snippet: STAT3 functions as a direct target of Visomitin to modulate LDHB transcription. (A) The potential transcription factors (TFs) for LDHB were predicted using the KnockTF, ENCODE, and ChIP_Atlas databases. (B) The potential targets of Visomitin were predicted using the SuperPRED database. (C and D) The mRNA and protein expression levels of LDHB under Stattic treatment ( n = 3). (E) The thermal stability of FLAG-STAT3 under Visomitin treatment was detected using WB ( n = 3). (F) The stability of FLAG-STAT3 in the presence of protease following treatment with Visomitin (0, 75, 150, and 300 nmol/l) was detected using WB ( n = 3). (G) Three-dimensional image of molecular docking between Visomitin and STAT3. (H) Representative immunoblots for the indicated nuclear, phosphorylated, or total proteins following treatment with Visomitin (0, 75, 150, and 300 nm) ( n = 3). (I) Representative Immunofluorescence images of STAT3 in BMMs after treatment with RANKL or Visomitin (0, 75, 150, and 300 nm) as indicated. Scale bars, 20 μm. (J) Quantification of Pearson’s correlation coefficient between STAT3 and DAPI in panel (I) ( n = 3). (K) STAT3 ChIP assay of LDHB promoter region. (L) Quantification of the binding affinity between STAT3 and the LDHB promoter ( n = 3). Data are mean ± SD; * P < 0.05, ** P < 0.01, and *** P < 0.001; ns, not significant.

Article Snippet: Recombinant soluble mouse macrophage colony-stimulating factor (M-CSF) (Cat. No. CB34) was supplied by Novoprotein; Recombinant soluble mouse RANKL (Cat. No. 462-TEC-010) were purchased from R&D Systems; Total Antioxidant Capacity (T-AOC) Assay Kit (Cat. No. S0121), ROS Assay Kit (Cat. No. S0033S), Mitochondrial membrane potential assay kit with JC-1 (Cat. No. C2006), MitoSOX Assay Kit (Cat. No. S0061S), DHE (Cat. No. S0063), and Antifade Mounting Medium (Cat. No. P0126) were supplied by Beyotime. iF488-wheat germ agglutinin (Cat. No. G1730) was purchased from Servicebio.

Techniques: Expressing, Western Blot, Immunofluorescence, Binding Assay