Journal: Scientific Reports

Article Title: Amlexanox Suppresses Osteoclastogenesis and Prevents Ovariectomy-Induced Bone Loss

doi: 10.1038/srep13575

Figure Lengend Snippet: ( a ) BMMs were seeded on day 0. RANKL was added on day 1 and every 2 days thereafter. Cells were collected for analysis of protein expression of TBK1 and IKK-ε. ACTB was used as a loading control. ( b ) Amlexanox has little effect on proliferation of BMMs. BMMs (5 × 10 3 cells/well) were cultured with M-CSF (30 ng/ml), amlexanox was added at different concentrations (0, 1.5, 3, 6, 12, 25 μM) every 2 days for 7 days. Cell proliferation was assessed by Cell Counting Kit-8. Data was presented as mean ± SD of 3 independent experiments. ( c,d ) Amlexanox inhibits osteoclast formation in a dose-dependent manner. BMMs (1 × 10 4 cells/well) were treated with different concentrations of amlexanox every 2 days in the presence of RANKL (50 ng/mL) and M-CSF (30 ng/ml) for 7 days. Then cells were fixed and stained for TRAP assay. TRAP-positive multinucleated osteoclasts (>=3 nuclei) were counted. Data was presented as mean ± SD of 3 independent experiments. Scale bar represents 100 μm. ( e,f ) BMMs (1 × 10 4 cells/well) were cultured in the presence of RANKL (50 ng/mL) and M-CSF (30 ng/ml) for 7 days and treated with 25 μM amlexanox at the indicated times. Subsequently, cells were photographed and TRAP-positive multinucleated osteoclasts (>=3 nuclei) were counted. Data was presented as mean ± SD of at least 3 independent experiments. *P < 0.05, **P < 0.01 versus vehicle. Scale bar represents 200 μm ( e ).

Article Snippet: Recombinant soluble human macrophage-colony stimulating factor (M-CSF) and mouse receptor activator of nuclear factor-κB ligand (RANKL) were obtained from Peprotech (Rocky Hill, NJ, USA).

Techniques: Expressing, Control, Cell Culture, Cell Counting, Staining, TRAP Assay

Journal: Scientific Reports

Article Title: Amlexanox Suppresses Osteoclastogenesis and Prevents Ovariectomy-Induced Bone Loss

doi: 10.1038/srep13575

Figure Lengend Snippet: ( a ) Amlexanox inhibits RANKL-induced phosphorylation of NF-κB/p65. We starved BMMs with 0.5% FBS in α-MEM for 12 h before treatment. BMMs were then pretreated with or without amlexanox (25 μM) for 1 h and then stimulated with RANKL (100 ng/mL) for the indicated times. The cell lysates were extracted for immunoblotting with the indicated antibodies. ( b ) Amlexanox suppresses RANKL-induced NF-κB DNA-binding activity. BMMs were pretreated with or without 25 μM amlexanox for 1 h and then stimulated with or without RANKL (100 ng/mL) for 30 min. Then the nuclear protein was prepared and subjected to EMSA. The Arrows indicate the free probe and the probe-NF-κB complex, respectively. ( c ) Amlexanox inhibits RANKL-induced phosphorylation of ERK, JNK and p38. We starved BMMs with 0.5% FBS in α-MEM for 12 h before treatment. BMMs were then pretreated with or without amlexanox (25 μM) for 1 h and then stimulated with RANKL (100 ng/mL) for the indicated times. The cell lysates were extracted for immunoblotting with the indicated antibodies.

Article Snippet: Recombinant soluble human macrophage-colony stimulating factor (M-CSF) and mouse receptor activator of nuclear factor-κB ligand (RANKL) were obtained from Peprotech (Rocky Hill, NJ, USA).

Techniques: Phospho-proteomics, Western Blot, Binding Assay, Activity Assay

Journal: Scientific Reports

Article Title: Amlexanox Suppresses Osteoclastogenesis and Prevents Ovariectomy-Induced Bone Loss

doi: 10.1038/srep13575

Figure Lengend Snippet: ( a ) Amlexanox inhibits RANKL-induced protein expression of c-Fos and NFATc1. BMMs were treated with or without amlexanox (25 μM) in the presence of RANKL (50 ng/mL) and M-CSF (30 ng/ml) for 7 days. Protein expression levels of c-Fos and NFATc1 were examined by western blot analysis at the indicated times. ( b,c ) Amlexanox suppresses RANKL-induced mRNA expression of TRAP, NFATc1, Cathepsin K, and MMP9. BMMs were treated with or without amlexanox (25 μM) in the presence of RANKL (50 ng/mL) and M-CSF (30 ng/ml) for 2 or 7 days. Total RNA was isolated and analyzed by quantitative real-time RT-PCR. Data represent as mean ± SD. *P < 0.05; **P < 0.01.

Article Snippet: Recombinant soluble human macrophage-colony stimulating factor (M-CSF) and mouse receptor activator of nuclear factor-κB ligand (RANKL) were obtained from Peprotech (Rocky Hill, NJ, USA).

Techniques: Expressing, Western Blot, Isolation, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: Amlexanox Suppresses Osteoclastogenesis and Prevents Ovariectomy-Induced Bone Loss

doi: 10.1038/srep13575

Figure Lengend Snippet: Mice were sham ovariectomized or ovariectomized as described, and treated with vehicle or amlexanox (20 mg/kg/d) for 8 weeks. Serum were collected before sacrifice. Serum levels of CTX-I ( a ) and osteocalcin ( b ) in SHAM, OVX and OVX+A mice were measured by ELISA. Data represent as mean ± SD. n = 12.*P < 0.05. ( c ) Serum levels of RANKL and OPG, and the RANKL: OPG ratio of animals was determined by ELISA. Data represent as mean ± SD. n = 12. *P < 0.05.

Article Snippet: Recombinant soluble human macrophage-colony stimulating factor (M-CSF) and mouse receptor activator of nuclear factor-κB ligand (RANKL) were obtained from Peprotech (Rocky Hill, NJ, USA).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Cell Reports Medicine

Article Title: CV1-secreting sCAR-T cells potentiate the abscopal effect of microwave ablation in heterogeneous tumors

doi: 10.1016/j.xcrm.2025.101965

Figure Lengend Snippet:

Article Snippet: Mouse M-CSF Recombinant Protein , PeproTech , Cat315-02-1MG.

Techniques: Recombinant, Protease Inhibitor, Selection, Cell Isolation, Enzyme-linked Immunosorbent Assay, RNA Extraction, Software

Journal: Bioactive Materials

Article Title: Immune regulative GelMA&Zn 2+ /Ce 3+ -whitlockite scaffolds with continuous ions release for bone regeneration

doi: 10.1016/j.bioactmat.2025.11.009

Figure Lengend Snippet: The Impact of Conditioned Media from RANKL-Induced Macrophages on Osteoclastogenesis In vitro . A) TRAP staining of RANKL-treated RAW264.7 cells co-cultured with different sample extracts. B) Measurement of TRAP enzyme activity at OD 405 nm. C) Phalloidin staining of RANKL-treated RAW264.7 cells co-cultured with different sample extracts. P values were determined using one-way ANOVA (∗∗∗P < 0.001, ∗∗P < 0.01, ∗P < 0.05, N.S., not significant).

Article Snippet: The conditioned medium for the RAW264.7 cells was supplemented with receptor activator of nuclear factor-κB ligand (RANKL) (Novoprotein Scientific Co., Ltd., China) to a final concentration of 100 ng/mL.

Techniques: In Vitro, Staining, Cell Culture, Activity Assay