Structured Review

Cell Signaling Technology Inc nfatc1
TRAIL inhibits RANKL-induced osteoclast differentiation and activation of <t>NFATc1.</t> a Bone marrow-derived macrophages (BMMs) from wild type (WT) and TRAIL-R knockout ( Trail-r −/− ) mice were plated in 96-well plates and stimulated with the RANKL (50 ng/ml) + M-CSF (20 ng/ml), TRAIL (500 ng/ml), or RANKL + M-CSF + TRAIL as indicated in the figure. After 10 days, cells were analyzed for osteoclast differentiation. After incubation, cells were subjected to a tartrate-resistant acid phosphatase (TRAP) assay. Cell morphology was examined by light microscopy (Scale bars, 100 µm), and the number of TRAP-positive multinuclear cells was quantified in ( b ). ** p
Nfatc1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/nfatc1/product/Cell Signaling Technology Inc
Average 99 stars, based on 7 article reviews
Price from $9.99 to $1999.99
nfatc1 - by Bioz Stars, 2020-09
99/100 stars

Images

1) Product Images from "TRAIL inhibits RANK signaling and suppresses osteoclast activation via inhibiting lipid raft assembly and TRAF6 recruitment"

Article Title: TRAIL inhibits RANK signaling and suppresses osteoclast activation via inhibiting lipid raft assembly and TRAF6 recruitment

Journal: Cell Death & Disease

doi: 10.1038/s41419-019-1353-3

TRAIL inhibits RANKL-induced osteoclast differentiation and activation of NFATc1. a Bone marrow-derived macrophages (BMMs) from wild type (WT) and TRAIL-R knockout ( Trail-r −/− ) mice were plated in 96-well plates and stimulated with the RANKL (50 ng/ml) + M-CSF (20 ng/ml), TRAIL (500 ng/ml), or RANKL + M-CSF + TRAIL as indicated in the figure. After 10 days, cells were analyzed for osteoclast differentiation. After incubation, cells were subjected to a tartrate-resistant acid phosphatase (TRAP) assay. Cell morphology was examined by light microscopy (Scale bars, 100 µm), and the number of TRAP-positive multinuclear cells was quantified in ( b ). ** p
Figure Legend Snippet: TRAIL inhibits RANKL-induced osteoclast differentiation and activation of NFATc1. a Bone marrow-derived macrophages (BMMs) from wild type (WT) and TRAIL-R knockout ( Trail-r −/− ) mice were plated in 96-well plates and stimulated with the RANKL (50 ng/ml) + M-CSF (20 ng/ml), TRAIL (500 ng/ml), or RANKL + M-CSF + TRAIL as indicated in the figure. After 10 days, cells were analyzed for osteoclast differentiation. After incubation, cells were subjected to a tartrate-resistant acid phosphatase (TRAP) assay. Cell morphology was examined by light microscopy (Scale bars, 100 µm), and the number of TRAP-positive multinuclear cells was quantified in ( b ). ** p

Techniques Used: Activation Assay, Derivative Assay, Knock-Out, Mouse Assay, Incubation, TRAP Assay, Light Microscopy

2) Product Images from "TRAIL inhibits RANK signaling and suppresses osteoclast activation via inhibiting lipid raft assembly and TRAF6 recruitment"

Article Title: TRAIL inhibits RANK signaling and suppresses osteoclast activation via inhibiting lipid raft assembly and TRAF6 recruitment

Journal: Cell Death & Disease

doi: 10.1038/s41419-019-1353-3

TRAIL inhibits RANKL-induced osteoclast differentiation and activation of NFATc1. a Bone marrow-derived macrophages (BMMs) from wild type (WT) and TRAIL-R knockout ( Trail-r −/− ) mice were plated in 96-well plates and stimulated with the RANKL (50 ng/ml) + M-CSF (20 ng/ml), TRAIL (500 ng/ml), or RANKL + M-CSF + TRAIL as indicated in the figure. After 10 days, cells were analyzed for osteoclast differentiation. After incubation, cells were subjected to a tartrate-resistant acid phosphatase (TRAP) assay. Cell morphology was examined by light microscopy (Scale bars, 100 µm), and the number of TRAP-positive multinuclear cells was quantified in ( b ). ** p
Figure Legend Snippet: TRAIL inhibits RANKL-induced osteoclast differentiation and activation of NFATc1. a Bone marrow-derived macrophages (BMMs) from wild type (WT) and TRAIL-R knockout ( Trail-r −/− ) mice were plated in 96-well plates and stimulated with the RANKL (50 ng/ml) + M-CSF (20 ng/ml), TRAIL (500 ng/ml), or RANKL + M-CSF + TRAIL as indicated in the figure. After 10 days, cells were analyzed for osteoclast differentiation. After incubation, cells were subjected to a tartrate-resistant acid phosphatase (TRAP) assay. Cell morphology was examined by light microscopy (Scale bars, 100 µm), and the number of TRAP-positive multinuclear cells was quantified in ( b ). ** p

Techniques Used: Activation Assay, Derivative Assay, Knock-Out, Mouse Assay, Incubation, TRAP Assay, Light Microscopy

3) Product Images from "Hypericin targets osteoclast and prevents breast cancer-induced bone metastasis via NFATc1 signaling pathway"

Article Title: Hypericin targets osteoclast and prevents breast cancer-induced bone metastasis via NFATc1 signaling pathway

Journal: Oncotarget

doi: 10.18632/oncotarget.22930

Hypericin inhibits breast cancer-induced osteoclast differentiation and function via suppression of the NFATc1 signaling pathway and attenuation of Ca 2+ oscillation in osteoclasts ( A ) RAW264.7 cells (3 × 10 3 cells/well) were incubated in the presence of MCF-7 or MDA-MB-231 cells for 24 h, exposed to HP (1 μmol/L) for 5 days, and finally stained for measurement of TRAP expression. ( B ) Multinucleated osteoclasts ( > 3 nuclei) in co-cultures were counted. Columns represent the mean results of experiments carried out in triplicate, whereas bars represent the SD. ( C ) HP-inhibited osteoclast bone resorption induced by tumor cells. RAW264.7 cells (3 × 10 3 cells/well) were seeded into bovine bone slices in the presence of MCF-7 or MDA-MB-231 cells for 24 h, and treated with HP (1 μmol/L) for 5 days. After 5 days of incubation, images were obtained using a scanning electron microscope (SEM). Images of bone resorption pits are shown. ( D ) Resorption pit areas were measured using ImageJ software. Columns represent the mean results of experiments carried out in triplicate, whereas bars represent the SD. ( E ) RAW264.7 cells were incubated in serum-free media containing the indicated concentrations of HP and RANKL for 24 h. The cells were lysed, and total RNA was subjected to RT-PCR for determination of NFATc1 gene expression. Graphs indicate the relative intensity of NFATc1 compared to that of GAPDH. ( F ) RAW264.7 cells that were stably transfected with a NFATc1 luciferase reporter construct were pretreated with the indicated concentrations of HP for 1 h and then incubated in the absence or presence of RANKL for 12 h. Luciferase activity was then determined using the Promega luciferase assay system. ( G ) RANKL-induced NFATc1 translocation to the nucleus was assessed by western blotting. RAW264.7 cells (1 × 10 6 cells/well) were pretreated with the indicated concentrations of HP for 2 h and then stimulated with RANKL (50 ng/mL) or were untreated (controls) for 15 min. Cell nuclear extracts were prepared and subjected to western blotting using anti-NFATc1 and LaminA/C. Cell cytosol extracts were prepared and subjected to western blotting using anti-phospho-NFATc1 and actin. ( H ) Effects of HP on nuclear translocation of NFATc1 in RAW264.7 cells. Cells were treated with RANKL for 72 h in the presence and absence of HP (1 mmol/L) and stained with anti-NFATc1 antibody to investigate NFATc1 nuclear translocation (left panel). Nuclei were stained with DAPI (middle panel). Merged images of NFATc1 and the nuclei are shown in the right panel. ( I ) HP reduces intracellular Ca 2+ levels and calcium influx. RAW264.7 cells (3 × 10 3 cells/well) were incubated with RANKL (100 ng/mL) in the presence or absence of HP (1 μM) for 72 h. For Ca 2+ measurement, cells were incubated with Fluo-4 AM and 0.05% pluronic F-127 (Invitrogen) in HBSS supplemented with 1% FCS and 1 mM probenecid (assay buffer) for 30 min followed by confocal analysis. Representative fluo-4 fluorescent images of the RAW264.7 cells from different treatment groups are shown. Pseudo-color-labeled (purple) area represents the cells that are actively undergoing fluorescence ratio changes. ( J ) The relative intracellular Ca 2+ levels in individual cells were monitored for 5 min at 5-second intervals using the fluorescence intensity of Fluo-4 at 200× magnification. Cells with at least two oscillations were counted as oscillating cells. A minimum of 40 cells were monitored in triplicate wells. The average amplitude of Ca 2+ oscillations in each cell was calculated using the TuneR and SeeWave packages for the R programming language. Representative traces of three randomly chosen BMMs were recorded in different treatment groups. The fluorescence ratio change was recorded every 5 s for 300 s.
Figure Legend Snippet: Hypericin inhibits breast cancer-induced osteoclast differentiation and function via suppression of the NFATc1 signaling pathway and attenuation of Ca 2+ oscillation in osteoclasts ( A ) RAW264.7 cells (3 × 10 3 cells/well) were incubated in the presence of MCF-7 or MDA-MB-231 cells for 24 h, exposed to HP (1 μmol/L) for 5 days, and finally stained for measurement of TRAP expression. ( B ) Multinucleated osteoclasts ( > 3 nuclei) in co-cultures were counted. Columns represent the mean results of experiments carried out in triplicate, whereas bars represent the SD. ( C ) HP-inhibited osteoclast bone resorption induced by tumor cells. RAW264.7 cells (3 × 10 3 cells/well) were seeded into bovine bone slices in the presence of MCF-7 or MDA-MB-231 cells for 24 h, and treated with HP (1 μmol/L) for 5 days. After 5 days of incubation, images were obtained using a scanning electron microscope (SEM). Images of bone resorption pits are shown. ( D ) Resorption pit areas were measured using ImageJ software. Columns represent the mean results of experiments carried out in triplicate, whereas bars represent the SD. ( E ) RAW264.7 cells were incubated in serum-free media containing the indicated concentrations of HP and RANKL for 24 h. The cells were lysed, and total RNA was subjected to RT-PCR for determination of NFATc1 gene expression. Graphs indicate the relative intensity of NFATc1 compared to that of GAPDH. ( F ) RAW264.7 cells that were stably transfected with a NFATc1 luciferase reporter construct were pretreated with the indicated concentrations of HP for 1 h and then incubated in the absence or presence of RANKL for 12 h. Luciferase activity was then determined using the Promega luciferase assay system. ( G ) RANKL-induced NFATc1 translocation to the nucleus was assessed by western blotting. RAW264.7 cells (1 × 10 6 cells/well) were pretreated with the indicated concentrations of HP for 2 h and then stimulated with RANKL (50 ng/mL) or were untreated (controls) for 15 min. Cell nuclear extracts were prepared and subjected to western blotting using anti-NFATc1 and LaminA/C. Cell cytosol extracts were prepared and subjected to western blotting using anti-phospho-NFATc1 and actin. ( H ) Effects of HP on nuclear translocation of NFATc1 in RAW264.7 cells. Cells were treated with RANKL for 72 h in the presence and absence of HP (1 mmol/L) and stained with anti-NFATc1 antibody to investigate NFATc1 nuclear translocation (left panel). Nuclei were stained with DAPI (middle panel). Merged images of NFATc1 and the nuclei are shown in the right panel. ( I ) HP reduces intracellular Ca 2+ levels and calcium influx. RAW264.7 cells (3 × 10 3 cells/well) were incubated with RANKL (100 ng/mL) in the presence or absence of HP (1 μM) for 72 h. For Ca 2+ measurement, cells were incubated with Fluo-4 AM and 0.05% pluronic F-127 (Invitrogen) in HBSS supplemented with 1% FCS and 1 mM probenecid (assay buffer) for 30 min followed by confocal analysis. Representative fluo-4 fluorescent images of the RAW264.7 cells from different treatment groups are shown. Pseudo-color-labeled (purple) area represents the cells that are actively undergoing fluorescence ratio changes. ( J ) The relative intracellular Ca 2+ levels in individual cells were monitored for 5 min at 5-second intervals using the fluorescence intensity of Fluo-4 at 200× magnification. Cells with at least two oscillations were counted as oscillating cells. A minimum of 40 cells were monitored in triplicate wells. The average amplitude of Ca 2+ oscillations in each cell was calculated using the TuneR and SeeWave packages for the R programming language. Representative traces of three randomly chosen BMMs were recorded in different treatment groups. The fluorescence ratio change was recorded every 5 s for 300 s.

Techniques Used: Incubation, Multiple Displacement Amplification, Staining, Expressing, Microscopy, Software, Reverse Transcription Polymerase Chain Reaction, Stable Transfection, Transfection, Luciferase, Construct, Activity Assay, Translocation Assay, Western Blot, Labeling, Fluorescence

4) Product Images from "Adenovirus-Mediated siRNA Targeting CXCR2 Attenuates Titanium Particle-Induced Osteolysis by Suppressing Osteoclast Formation"

Article Title: Adenovirus-Mediated siRNA Targeting CXCR2 Attenuates Titanium Particle-Induced Osteolysis by Suppressing Osteoclast Formation

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

doi: 10.12659/MSM.897243

Adenovirus-mediated CXCR2 siRNA inhibited RANKL-induced osteoclast-specific gene and protein expression in vitro . ( A ) The mRNA expression of RANK , TRAP , CtsK , and c-Fos was significantly decreased in the CXCR2 group compared with the MS and control groups. ( B ) Western blotting revealed that TRAP, CtsK, and NFATc1 levels were decreased in the CXCR2 group. There were no other significant differences between the MS and control groups. All experiments were performed at least 3 times. * P
Figure Legend Snippet: Adenovirus-mediated CXCR2 siRNA inhibited RANKL-induced osteoclast-specific gene and protein expression in vitro . ( A ) The mRNA expression of RANK , TRAP , CtsK , and c-Fos was significantly decreased in the CXCR2 group compared with the MS and control groups. ( B ) Western blotting revealed that TRAP, CtsK, and NFATc1 levels were decreased in the CXCR2 group. There were no other significant differences between the MS and control groups. All experiments were performed at least 3 times. * P

Techniques Used: Expressing, In Vitro, Mass Spectrometry, Western Blot

5) Product Images from "TRAIL inhibits RANK signaling and suppresses osteoclast activation via inhibiting lipid raft assembly and TRAF6 recruitment"

Article Title: TRAIL inhibits RANK signaling and suppresses osteoclast activation via inhibiting lipid raft assembly and TRAF6 recruitment

Journal: Cell Death & Disease

doi: 10.1038/s41419-019-1353-3

TRAIL inhibits RANKL-induced osteoclast differentiation and activation of NFATc1. a Bone marrow-derived macrophages (BMMs) from wild type (WT) and TRAIL-R knockout ( Trail-r −/− ) mice were plated in 96-well plates and stimulated with the RANKL (50 ng/ml) + M-CSF (20 ng/ml), TRAIL (500 ng/ml), or RANKL + M-CSF + TRAIL as indicated in the figure. After 10 days, cells were analyzed for osteoclast differentiation. After incubation, cells were subjected to a tartrate-resistant acid phosphatase (TRAP) assay. Cell morphology was examined by light microscopy (Scale bars, 100 µm), and the number of TRAP-positive multinuclear cells was quantified in ( b ). ** p
Figure Legend Snippet: TRAIL inhibits RANKL-induced osteoclast differentiation and activation of NFATc1. a Bone marrow-derived macrophages (BMMs) from wild type (WT) and TRAIL-R knockout ( Trail-r −/− ) mice were plated in 96-well plates and stimulated with the RANKL (50 ng/ml) + M-CSF (20 ng/ml), TRAIL (500 ng/ml), or RANKL + M-CSF + TRAIL as indicated in the figure. After 10 days, cells were analyzed for osteoclast differentiation. After incubation, cells were subjected to a tartrate-resistant acid phosphatase (TRAP) assay. Cell morphology was examined by light microscopy (Scale bars, 100 µm), and the number of TRAP-positive multinuclear cells was quantified in ( b ). ** p

Techniques Used: Activation Assay, Derivative Assay, Knock-Out, Mouse Assay, Incubation, TRAP Assay, Light Microscopy

6) Product Images from "Isosteviol Derivative Inhibits Osteoclast Differentiation and Ameliorates Ovariectomy-Induced Osteoporosis"

Article Title: Isosteviol Derivative Inhibits Osteoclast Differentiation and Ameliorates Ovariectomy-Induced Osteoporosis

Journal: Scientific Reports

doi: 10.1038/s41598-018-29257-1

The inhibitory effects of NC-8 on the RANKL-induced osteoclast differentiation signaling pathway. ( A and B ) RAW 264.7 cells were treated with 100 ng/ml RANKL or with 10 μg/ml NC-8, and the protein was collected after the reaction. Western blot was used to detect MAPK pathway and downstream NFATc1, c-Fos and NF-κB protein molecules. ( A ) Relative protein expressions analysis of proteins related to the MAPK signaling pathway, including p-ERK, p-JNK, and p-p38. β-actin was used as a loading control. ( B ) Relative protein expressions analysis of proteins related to the NF-κB signaling pathway, including p65, NFATc1 and c-Fos. β-actin was used as a loading control. Relative expression levels of p65, NFATc1 and c-Fos protein in the cytoplasm and in the nuclear fractions. β-actin and HDAC1 were used as loading controls. The results are expressed as the mean ± S.E.M. of four independent experiments. *p
Figure Legend Snippet: The inhibitory effects of NC-8 on the RANKL-induced osteoclast differentiation signaling pathway. ( A and B ) RAW 264.7 cells were treated with 100 ng/ml RANKL or with 10 μg/ml NC-8, and the protein was collected after the reaction. Western blot was used to detect MAPK pathway and downstream NFATc1, c-Fos and NF-κB protein molecules. ( A ) Relative protein expressions analysis of proteins related to the MAPK signaling pathway, including p-ERK, p-JNK, and p-p38. β-actin was used as a loading control. ( B ) Relative protein expressions analysis of proteins related to the NF-κB signaling pathway, including p65, NFATc1 and c-Fos. β-actin was used as a loading control. Relative expression levels of p65, NFATc1 and c-Fos protein in the cytoplasm and in the nuclear fractions. β-actin and HDAC1 were used as loading controls. The results are expressed as the mean ± S.E.M. of four independent experiments. *p

Techniques Used: Western Blot, Expressing

7) Product Images from "Curcumin has immunomodulatory effects on RANKL‐stimulated osteoclastogenesis in vitro and titanium nanoparticle‐induced bone loss in vivo. Curcumin has immunomodulatory effects on RANKL‐stimulated osteoclastogenesis in vitro and titanium nanoparticle‐induced bone loss in vivo"

Article Title: Curcumin has immunomodulatory effects on RANKL‐stimulated osteoclastogenesis in vitro and titanium nanoparticle‐induced bone loss in vivo. Curcumin has immunomodulatory effects on RANKL‐stimulated osteoclastogenesis in vitro and titanium nanoparticle‐induced bone loss in vivo

Journal: Journal of Cellular and Molecular Medicine

doi: 10.1111/jcmm.14842

Curcumin ameliorated the activation of Akt and NF‐κB p65 phosphorylation but had no inhibitory effect on the MAPK pathways. (A and B), RAW264.7 cells were pretreated with or without curcumin for 4 h and then with 100 ng/mL RANKL for indicated time periods (0, 5, 15 or 30 min). Then, the cells were collected and lysed for Western blot analysis. C, The relative grey levels corresponding to p‐ERK, p‐JNK and p‐p38 were quantified and were normalized to β‐actin using ImageJ software. D, The relative grey levels corresponding to p‐Akt, p‐p65 and p‐IκBα were quantified and normalized to β‐actin using ImageJ software. E, RAW264.7 cells were pretreated with or without 20 μmol/L curcumin for 4 h and then stimulated by RANKL for 30 min. The cells were prepared for immunofluorescence staining of p65. F, RAW264.7 cells were cultured in osteoclast induction medium with or without 20 μmol/L curcumin for 1, 3 or 5 d. Cells were then collected and lysed for Western blot analysis. G, The relative grey levels corresponding to c‐fos and NFATc1 were quantified and normalized to β‐actin using ImageJ software. Data are presented as mean ± SD; * P
Figure Legend Snippet: Curcumin ameliorated the activation of Akt and NF‐κB p65 phosphorylation but had no inhibitory effect on the MAPK pathways. (A and B), RAW264.7 cells were pretreated with or without curcumin for 4 h and then with 100 ng/mL RANKL for indicated time periods (0, 5, 15 or 30 min). Then, the cells were collected and lysed for Western blot analysis. C, The relative grey levels corresponding to p‐ERK, p‐JNK and p‐p38 were quantified and were normalized to β‐actin using ImageJ software. D, The relative grey levels corresponding to p‐Akt, p‐p65 and p‐IκBα were quantified and normalized to β‐actin using ImageJ software. E, RAW264.7 cells were pretreated with or without 20 μmol/L curcumin for 4 h and then stimulated by RANKL for 30 min. The cells were prepared for immunofluorescence staining of p65. F, RAW264.7 cells were cultured in osteoclast induction medium with or without 20 μmol/L curcumin for 1, 3 or 5 d. Cells were then collected and lysed for Western blot analysis. G, The relative grey levels corresponding to c‐fos and NFATc1 were quantified and normalized to β‐actin using ImageJ software. Data are presented as mean ± SD; * P

Techniques Used: Activation Assay, Western Blot, Software, Immunofluorescence, Staining, Cell Culture

Schematic illustration of curcumin has immunomodulatory and inhibitory effects on RANKL‐induced osteoclast formation. Curcumin attenuated the up‐regulation of Akt and NF‐κB p65 phosphorylation and the activation of the downstream transcription factor NFATc1. In addition, curcumin created an immunomodulatory microenvironment and promoted macrophage polarization from the M1‐type to the M2‐type phenotype
Figure Legend Snippet: Schematic illustration of curcumin has immunomodulatory and inhibitory effects on RANKL‐induced osteoclast formation. Curcumin attenuated the up‐regulation of Akt and NF‐κB p65 phosphorylation and the activation of the downstream transcription factor NFATc1. In addition, curcumin created an immunomodulatory microenvironment and promoted macrophage polarization from the M1‐type to the M2‐type phenotype

Techniques Used: Activation Assay

Curcumin down‐regulated the expression of RANKL‐induced osteoclast‐related genes including NFATc1, c‐fos, Oscar and Sema‐4A in vitro. A, BMMs were cultured and induced in osteoclast induction medium containing various curcumin concentrations (0, 1.25, 5 or 20 μmol/L) for 5 d. B, BMMs were incubated in osteoclast induction medium with or without 20 μmol/L curcumin for 1, 3 or 5 d. The expression of osteoclast‐related genes was quantified by RT‐PCR. Data are presented as mean ± SD; * P
Figure Legend Snippet: Curcumin down‐regulated the expression of RANKL‐induced osteoclast‐related genes including NFATc1, c‐fos, Oscar and Sema‐4A in vitro. A, BMMs were cultured and induced in osteoclast induction medium containing various curcumin concentrations (0, 1.25, 5 or 20 μmol/L) for 5 d. B, BMMs were incubated in osteoclast induction medium with or without 20 μmol/L curcumin for 1, 3 or 5 d. The expression of osteoclast‐related genes was quantified by RT‐PCR. Data are presented as mean ± SD; * P

Techniques Used: Expressing, In Vitro, Cell Culture, Incubation, Reverse Transcription Polymerase Chain Reaction

8) Product Images from "Curcumin has immunomodulatory effects on RANKL‐stimulated osteoclastogenesis in vitro and titanium nanoparticle‐induced bone loss in vivo. Curcumin has immunomodulatory effects on RANKL‐stimulated osteoclastogenesis in vitro and titanium nanoparticle‐induced bone loss in vivo"

Article Title: Curcumin has immunomodulatory effects on RANKL‐stimulated osteoclastogenesis in vitro and titanium nanoparticle‐induced bone loss in vivo. Curcumin has immunomodulatory effects on RANKL‐stimulated osteoclastogenesis in vitro and titanium nanoparticle‐induced bone loss in vivo

Journal: Journal of Cellular and Molecular Medicine

doi: 10.1111/jcmm.14842

Curcumin ameliorated the activation of Akt and NF‐κB p65 phosphorylation but had no inhibitory effect on the MAPK pathways. (A and B), RAW264.7 cells were pretreated with or without curcumin for 4 h and then with 100 ng/mL RANKL for indicated time periods (0, 5, 15 or 30 min). Then, the cells were collected and lysed for Western blot analysis. C, The relative grey levels corresponding to p‐ERK, p‐JNK and p‐p38 were quantified and were normalized to β‐actin using ImageJ software. D, The relative grey levels corresponding to p‐Akt, p‐p65 and p‐IκBα were quantified and normalized to β‐actin using ImageJ software. E, RAW264.7 cells were pretreated with or without 20 μmol/L curcumin for 4 h and then stimulated by RANKL for 30 min. The cells were prepared for immunofluorescence staining of p65. F, RAW264.7 cells were cultured in osteoclast induction medium with or without 20 μmol/L curcumin for 1, 3 or 5 d. Cells were then collected and lysed for Western blot analysis. G, The relative grey levels corresponding to c‐fos and NFATc1 were quantified and normalized to β‐actin using ImageJ software. Data are presented as mean ± SD; * P
Figure Legend Snippet: Curcumin ameliorated the activation of Akt and NF‐κB p65 phosphorylation but had no inhibitory effect on the MAPK pathways. (A and B), RAW264.7 cells were pretreated with or without curcumin for 4 h and then with 100 ng/mL RANKL for indicated time periods (0, 5, 15 or 30 min). Then, the cells were collected and lysed for Western blot analysis. C, The relative grey levels corresponding to p‐ERK, p‐JNK and p‐p38 were quantified and were normalized to β‐actin using ImageJ software. D, The relative grey levels corresponding to p‐Akt, p‐p65 and p‐IκBα were quantified and normalized to β‐actin using ImageJ software. E, RAW264.7 cells were pretreated with or without 20 μmol/L curcumin for 4 h and then stimulated by RANKL for 30 min. The cells were prepared for immunofluorescence staining of p65. F, RAW264.7 cells were cultured in osteoclast induction medium with or without 20 μmol/L curcumin for 1, 3 or 5 d. Cells were then collected and lysed for Western blot analysis. G, The relative grey levels corresponding to c‐fos and NFATc1 were quantified and normalized to β‐actin using ImageJ software. Data are presented as mean ± SD; * P

Techniques Used: Activation Assay, Western Blot, Software, Immunofluorescence, Staining, Cell Culture

Schematic illustration of curcumin has immunomodulatory and inhibitory effects on RANKL‐induced osteoclast formation. Curcumin attenuated the up‐regulation of Akt and NF‐κB p65 phosphorylation and the activation of the downstream transcription factor NFATc1. In addition, curcumin created an immunomodulatory microenvironment and promoted macrophage polarization from the M1‐type to the M2‐type phenotype
Figure Legend Snippet: Schematic illustration of curcumin has immunomodulatory and inhibitory effects on RANKL‐induced osteoclast formation. Curcumin attenuated the up‐regulation of Akt and NF‐κB p65 phosphorylation and the activation of the downstream transcription factor NFATc1. In addition, curcumin created an immunomodulatory microenvironment and promoted macrophage polarization from the M1‐type to the M2‐type phenotype

Techniques Used: Activation Assay

Curcumin down‐regulated the expression of RANKL‐induced osteoclast‐related genes including NFATc1, c‐fos, Oscar and Sema‐4A in vitro. A, BMMs were cultured and induced in osteoclast induction medium containing various curcumin concentrations (0, 1.25, 5 or 20 μmol/L) for 5 d. B, BMMs were incubated in osteoclast induction medium with or without 20 μmol/L curcumin for 1, 3 or 5 d. The expression of osteoclast‐related genes was quantified by RT‐PCR. Data are presented as mean ± SD; * P
Figure Legend Snippet: Curcumin down‐regulated the expression of RANKL‐induced osteoclast‐related genes including NFATc1, c‐fos, Oscar and Sema‐4A in vitro. A, BMMs were cultured and induced in osteoclast induction medium containing various curcumin concentrations (0, 1.25, 5 or 20 μmol/L) for 5 d. B, BMMs were incubated in osteoclast induction medium with or without 20 μmol/L curcumin for 1, 3 or 5 d. The expression of osteoclast‐related genes was quantified by RT‐PCR. Data are presented as mean ± SD; * P

Techniques Used: Expressing, In Vitro, Cell Culture, Incubation, Reverse Transcription Polymerase Chain Reaction

9) Product Images from "Curcumin has immunomodulatory effects on RANKL‐stimulated osteoclastogenesis in vitro and titanium nanoparticle‐induced bone loss in vivo. Curcumin has immunomodulatory effects on RANKL‐stimulated osteoclastogenesis in vitro and titanium nanoparticle‐induced bone loss in vivo"

Article Title: Curcumin has immunomodulatory effects on RANKL‐stimulated osteoclastogenesis in vitro and titanium nanoparticle‐induced bone loss in vivo. Curcumin has immunomodulatory effects on RANKL‐stimulated osteoclastogenesis in vitro and titanium nanoparticle‐induced bone loss in vivo

Journal: Journal of Cellular and Molecular Medicine

doi: 10.1111/jcmm.14842

Curcumin ameliorated the activation of Akt and NF‐κB p65 phosphorylation but had no inhibitory effect on the MAPK pathways. (A and B), RAW264.7 cells were pretreated with or without curcumin for 4 h and then with 100 ng/mL RANKL for indicated time periods (0, 5, 15 or 30 min). Then, the cells were collected and lysed for Western blot analysis. C, The relative grey levels corresponding to p‐ERK, p‐JNK and p‐p38 were quantified and were normalized to β‐actin using ImageJ software. D, The relative grey levels corresponding to p‐Akt, p‐p65 and p‐IκBα were quantified and normalized to β‐actin using ImageJ software. E, RAW264.7 cells were pretreated with or without 20 μmol/L curcumin for 4 h and then stimulated by RANKL for 30 min. The cells were prepared for immunofluorescence staining of p65. F, RAW264.7 cells were cultured in osteoclast induction medium with or without 20 μmol/L curcumin for 1, 3 or 5 d. Cells were then collected and lysed for Western blot analysis. G, The relative grey levels corresponding to c‐fos and NFATc1 were quantified and normalized to β‐actin using ImageJ software. Data are presented as mean ± SD; * P
Figure Legend Snippet: Curcumin ameliorated the activation of Akt and NF‐κB p65 phosphorylation but had no inhibitory effect on the MAPK pathways. (A and B), RAW264.7 cells were pretreated with or without curcumin for 4 h and then with 100 ng/mL RANKL for indicated time periods (0, 5, 15 or 30 min). Then, the cells were collected and lysed for Western blot analysis. C, The relative grey levels corresponding to p‐ERK, p‐JNK and p‐p38 were quantified and were normalized to β‐actin using ImageJ software. D, The relative grey levels corresponding to p‐Akt, p‐p65 and p‐IκBα were quantified and normalized to β‐actin using ImageJ software. E, RAW264.7 cells were pretreated with or without 20 μmol/L curcumin for 4 h and then stimulated by RANKL for 30 min. The cells were prepared for immunofluorescence staining of p65. F, RAW264.7 cells were cultured in osteoclast induction medium with or without 20 μmol/L curcumin for 1, 3 or 5 d. Cells were then collected and lysed for Western blot analysis. G, The relative grey levels corresponding to c‐fos and NFATc1 were quantified and normalized to β‐actin using ImageJ software. Data are presented as mean ± SD; * P

Techniques Used: Activation Assay, Western Blot, Software, Immunofluorescence, Staining, Cell Culture

Schematic illustration of curcumin has immunomodulatory and inhibitory effects on RANKL‐induced osteoclast formation. Curcumin attenuated the up‐regulation of Akt and NF‐κB p65 phosphorylation and the activation of the downstream transcription factor NFATc1. In addition, curcumin created an immunomodulatory microenvironment and promoted macrophage polarization from the M1‐type to the M2‐type phenotype
Figure Legend Snippet: Schematic illustration of curcumin has immunomodulatory and inhibitory effects on RANKL‐induced osteoclast formation. Curcumin attenuated the up‐regulation of Akt and NF‐κB p65 phosphorylation and the activation of the downstream transcription factor NFATc1. In addition, curcumin created an immunomodulatory microenvironment and promoted macrophage polarization from the M1‐type to the M2‐type phenotype

Techniques Used: Activation Assay

Curcumin down‐regulated the expression of RANKL‐induced osteoclast‐related genes including NFATc1, c‐fos, Oscar and Sema‐4A in vitro. A, BMMs were cultured and induced in osteoclast induction medium containing various curcumin concentrations (0, 1.25, 5 or 20 μmol/L) for 5 d. B, BMMs were incubated in osteoclast induction medium with or without 20 μmol/L curcumin for 1, 3 or 5 d. The expression of osteoclast‐related genes was quantified by RT‐PCR. Data are presented as mean ± SD; * P
Figure Legend Snippet: Curcumin down‐regulated the expression of RANKL‐induced osteoclast‐related genes including NFATc1, c‐fos, Oscar and Sema‐4A in vitro. A, BMMs were cultured and induced in osteoclast induction medium containing various curcumin concentrations (0, 1.25, 5 or 20 μmol/L) for 5 d. B, BMMs were incubated in osteoclast induction medium with or without 20 μmol/L curcumin for 1, 3 or 5 d. The expression of osteoclast‐related genes was quantified by RT‐PCR. Data are presented as mean ± SD; * P

Techniques Used: Expressing, In Vitro, Cell Culture, Incubation, Reverse Transcription Polymerase Chain Reaction

10) Product Images from "TRAIL inhibits RANK signaling and suppresses osteoclast activation via inhibiting lipid raft assembly and TRAF6 recruitment"

Article Title: TRAIL inhibits RANK signaling and suppresses osteoclast activation via inhibiting lipid raft assembly and TRAF6 recruitment

Journal: Cell Death & Disease

doi: 10.1038/s41419-019-1353-3

TRAIL inhibits RANKL-induced osteoclast differentiation and activation of NFATc1. a Bone marrow-derived macrophages (BMMs) from wild type (WT) and TRAIL-R knockout ( Trail-r −/− ) mice were plated in 96-well plates and stimulated with the RANKL (50 ng/ml) + M-CSF (20 ng/ml), TRAIL (500 ng/ml), or RANKL + M-CSF + TRAIL as indicated in the figure. After 10 days, cells were analyzed for osteoclast differentiation. After incubation, cells were subjected to a tartrate-resistant acid phosphatase (TRAP) assay. Cell morphology was examined by light microscopy (Scale bars, 100 µm), and the number of TRAP-positive multinuclear cells was quantified in ( b ). ** p
Figure Legend Snippet: TRAIL inhibits RANKL-induced osteoclast differentiation and activation of NFATc1. a Bone marrow-derived macrophages (BMMs) from wild type (WT) and TRAIL-R knockout ( Trail-r −/− ) mice were plated in 96-well plates and stimulated with the RANKL (50 ng/ml) + M-CSF (20 ng/ml), TRAIL (500 ng/ml), or RANKL + M-CSF + TRAIL as indicated in the figure. After 10 days, cells were analyzed for osteoclast differentiation. After incubation, cells were subjected to a tartrate-resistant acid phosphatase (TRAP) assay. Cell morphology was examined by light microscopy (Scale bars, 100 µm), and the number of TRAP-positive multinuclear cells was quantified in ( b ). ** p

Techniques Used: Activation Assay, Derivative Assay, Knock-Out, Mouse Assay, Incubation, TRAP Assay, Light Microscopy

11) Product Images from "TRAIL inhibits RANK signaling and suppresses osteoclast activation via inhibiting lipid raft assembly and TRAF6 recruitment"

Article Title: TRAIL inhibits RANK signaling and suppresses osteoclast activation via inhibiting lipid raft assembly and TRAF6 recruitment

Journal: Cell Death & Disease

doi: 10.1038/s41419-019-1353-3

TRAIL inhibits RANKL-induced osteoclast differentiation and activation of NFATc1. a Bone marrow-derived macrophages (BMMs) from wild type (WT) and TRAIL-R knockout ( Trail-r −/− ) mice were plated in 96-well plates and stimulated with the RANKL (50 ng/ml) + M-CSF (20 ng/ml), TRAIL (500 ng/ml), or RANKL + M-CSF + TRAIL as indicated in the figure. After 10 days, cells were analyzed for osteoclast differentiation. After incubation, cells were subjected to a tartrate-resistant acid phosphatase (TRAP) assay. Cell morphology was examined by light microscopy (Scale bars, 100 µm), and the number of TRAP-positive multinuclear cells was quantified in ( b ). ** p
Figure Legend Snippet: TRAIL inhibits RANKL-induced osteoclast differentiation and activation of NFATc1. a Bone marrow-derived macrophages (BMMs) from wild type (WT) and TRAIL-R knockout ( Trail-r −/− ) mice were plated in 96-well plates and stimulated with the RANKL (50 ng/ml) + M-CSF (20 ng/ml), TRAIL (500 ng/ml), or RANKL + M-CSF + TRAIL as indicated in the figure. After 10 days, cells were analyzed for osteoclast differentiation. After incubation, cells were subjected to a tartrate-resistant acid phosphatase (TRAP) assay. Cell morphology was examined by light microscopy (Scale bars, 100 µm), and the number of TRAP-positive multinuclear cells was quantified in ( b ). ** p

Techniques Used: Activation Assay, Derivative Assay, Knock-Out, Mouse Assay, Incubation, TRAP Assay, Light Microscopy

12) Product Images from "Inhibitory Effect of Cudratrixanthone U on RANKL-Induced Osteoclast Differentiation and Function in Macrophages and BMM Cells"

Article Title: Inhibitory Effect of Cudratrixanthone U on RANKL-Induced Osteoclast Differentiation and Function in Macrophages and BMM Cells

Journal: Frontiers in Pharmacology

doi: 10.3389/fphar.2020.01048

The effects of CTU on NFATc1 and c-Fos protein expression in RANKL-induced RAW 264.7 (A) and BMM (B) cells. The cells were cultured in the presence of RANKL with the CTU. After 24 h, the total protein was isolated and the protein expression levels were evaluated by western blots.* p
Figure Legend Snippet: The effects of CTU on NFATc1 and c-Fos protein expression in RANKL-induced RAW 264.7 (A) and BMM (B) cells. The cells were cultured in the presence of RANKL with the CTU. After 24 h, the total protein was isolated and the protein expression levels were evaluated by western blots.* p

Techniques Used: Expressing, Cell Culture, Isolation, Western Blot

13) Product Images from "CD44 deficiency inhibits unloading-induced cortical bone loss through downregulation of osteoclast activity"

Article Title: CD44 deficiency inhibits unloading-induced cortical bone loss through downregulation of osteoclast activity

Journal: Scientific Reports

doi: 10.1038/srep16124

CD44 regulates osteoclast differentiation through NF-κB signaling pathway. BMMs from two-month-old WT and CD44 KO mice were cultured with M-CSF (30 ng/ml) and RANKL (50 ng/ml) for 1, 3, and 5 days. Cell lysates were subjected to western blot analysis using specific antibodies. Representative western blot of p-Src (Tyr416) and c-Src ( A ), p-Akt (S473) and Akt ( B ), p-IκBα, IκBα and NF-κB ( C ), NFATc1 ( D ) were shown. GAPDH was used as internal control. ( E ) The effect of CD44 on the interaction between TRAF6 and RANK. BMMs from two-month-old WT and CD44 KO mice were cultured with M-CSF (30 ng/ml) and RANKL (50 ng/ml) for 5 days, the cell lysate was immunoprecipitated by TRAF6 antibody, followd by RANK detection with anti-RANK antibody.
Figure Legend Snippet: CD44 regulates osteoclast differentiation through NF-κB signaling pathway. BMMs from two-month-old WT and CD44 KO mice were cultured with M-CSF (30 ng/ml) and RANKL (50 ng/ml) for 1, 3, and 5 days. Cell lysates were subjected to western blot analysis using specific antibodies. Representative western blot of p-Src (Tyr416) and c-Src ( A ), p-Akt (S473) and Akt ( B ), p-IκBα, IκBα and NF-κB ( C ), NFATc1 ( D ) were shown. GAPDH was used as internal control. ( E ) The effect of CD44 on the interaction between TRAF6 and RANK. BMMs from two-month-old WT and CD44 KO mice were cultured with M-CSF (30 ng/ml) and RANKL (50 ng/ml) for 5 days, the cell lysate was immunoprecipitated by TRAF6 antibody, followd by RANK detection with anti-RANK antibody.

Techniques Used: Mouse Assay, Cell Culture, Western Blot, Immunoprecipitation

Model of CD44-mediated pathway in osteoclast differentiation and activity. After RANKL stimulation, the CD44 expression in osteoclast cells was upregulated. CD44 could increase the interaction between RANK and TRAF6, then it would activate its downstream signaling molecules, lead the phosphorylation of Src or Akt, which phosphorylates IκB-α and promotes the expression of NFATc1. NFATc1 induces the expression of genes related to the function and activity of osteoclast.
Figure Legend Snippet: Model of CD44-mediated pathway in osteoclast differentiation and activity. After RANKL stimulation, the CD44 expression in osteoclast cells was upregulated. CD44 could increase the interaction between RANK and TRAF6, then it would activate its downstream signaling molecules, lead the phosphorylation of Src or Akt, which phosphorylates IκB-α and promotes the expression of NFATc1. NFATc1 induces the expression of genes related to the function and activity of osteoclast.

Techniques Used: Activity Assay, Expressing

14) Product Images from "TRAIL inhibits RANK signaling and suppresses osteoclast activation via inhibiting lipid raft assembly and TRAF6 recruitment"

Article Title: TRAIL inhibits RANK signaling and suppresses osteoclast activation via inhibiting lipid raft assembly and TRAF6 recruitment

Journal: Cell Death & Disease

doi: 10.1038/s41419-019-1353-3

TRAIL inhibits RANKL-induced osteoclast differentiation and activation of NFATc1. a Bone marrow-derived macrophages (BMMs) from wild type (WT) and TRAIL-R knockout ( Trail-r −/− ) mice were plated in 96-well plates and stimulated with the RANKL (50 ng/ml) + M-CSF (20 ng/ml), TRAIL (500 ng/ml), or RANKL + M-CSF + TRAIL as indicated in the figure. After 10 days, cells were analyzed for osteoclast differentiation. After incubation, cells were subjected to a tartrate-resistant acid phosphatase (TRAP) assay. Cell morphology was examined by light microscopy (Scale bars, 100 µm), and the number of TRAP-positive multinuclear cells was quantified in ( b ). ** p
Figure Legend Snippet: TRAIL inhibits RANKL-induced osteoclast differentiation and activation of NFATc1. a Bone marrow-derived macrophages (BMMs) from wild type (WT) and TRAIL-R knockout ( Trail-r −/− ) mice were plated in 96-well plates and stimulated with the RANKL (50 ng/ml) + M-CSF (20 ng/ml), TRAIL (500 ng/ml), or RANKL + M-CSF + TRAIL as indicated in the figure. After 10 days, cells were analyzed for osteoclast differentiation. After incubation, cells were subjected to a tartrate-resistant acid phosphatase (TRAP) assay. Cell morphology was examined by light microscopy (Scale bars, 100 µm), and the number of TRAP-positive multinuclear cells was quantified in ( b ). ** p

Techniques Used: Activation Assay, Derivative Assay, Knock-Out, Mouse Assay, Incubation, TRAP Assay, Light Microscopy

15) Product Images from "Dendritic cells-derived interferon-λ1 ameliorated inflammatory bone destruction through inhibiting osteoclastogenesis"

Article Title: Dendritic cells-derived interferon-λ1 ameliorated inflammatory bone destruction through inhibiting osteoclastogenesis

Journal: Cell Death & Disease

doi: 10.1038/s41419-020-2612-z

IFN-λ1 inhibited the nuclei translocation of NFATc1 and the expression of osteoclast-specific genes. a RAW264.7 cells were seeded in 96-well plates and treated with IFN-λ1 (100 ng/ml) for 24 h, followed by stimulation with 100 ng/ml RANKL and 50 ng/ml M-CSF. The intracellular location of the NFATc1 was observed by immunofluorescence staining using confocal microscopy. Scale bar = 800 μm. b The gray values of the Green and Blue staining were measured using the Image J software, and the mean values were plotted using excel. c Relative mRNA expression of CD9, c-Fos, Ctsk, PU.1, and NFATc1 during treatment with RANKL in the presence or absence of IFN-λ1 (100 ng/ml) for 24 h. d Relative expression of c-Fos and NFATc1 during treatment with RANKL in the presence or absence of IFN-λ1 (100 ng/ml) for 24 h in protein level. β-actin was used as an internal control. e Relative expression of CD9, MMP-9, CTSK, c-Fos, and NFATc1 during treatment with RANKL or LPS in the presence or absence of IFN-λ1 (100 ng/ml) for 72 h in protein level. β-actin was used as an internal control. f Relative mRNA expression of mitf, c-Fos, Ctsk, CTR, and NFATc1 during treatment with RANKL in the presence or absence of IFN-λ1 (100 ng/ml) for 72 h. g Relative mRNA expression of mitf, Ctsk, CTR, and OC-STAMP during treatment with LPS in the presence or absence of IFN-λ1 (100 ng/ml) for 72 h. h Relative mRNA expression of IL-1β, IL-6, and TNF-α during LPS-induced osteoclastogenesis in the presence or absence of IFN-λ1 (100 ng/ml). The data in the figures represent the averages ± SD. Significant differences are indicated as * p
Figure Legend Snippet: IFN-λ1 inhibited the nuclei translocation of NFATc1 and the expression of osteoclast-specific genes. a RAW264.7 cells were seeded in 96-well plates and treated with IFN-λ1 (100 ng/ml) for 24 h, followed by stimulation with 100 ng/ml RANKL and 50 ng/ml M-CSF. The intracellular location of the NFATc1 was observed by immunofluorescence staining using confocal microscopy. Scale bar = 800 μm. b The gray values of the Green and Blue staining were measured using the Image J software, and the mean values were plotted using excel. c Relative mRNA expression of CD9, c-Fos, Ctsk, PU.1, and NFATc1 during treatment with RANKL in the presence or absence of IFN-λ1 (100 ng/ml) for 24 h. d Relative expression of c-Fos and NFATc1 during treatment with RANKL in the presence or absence of IFN-λ1 (100 ng/ml) for 24 h in protein level. β-actin was used as an internal control. e Relative expression of CD9, MMP-9, CTSK, c-Fos, and NFATc1 during treatment with RANKL or LPS in the presence or absence of IFN-λ1 (100 ng/ml) for 72 h in protein level. β-actin was used as an internal control. f Relative mRNA expression of mitf, c-Fos, Ctsk, CTR, and NFATc1 during treatment with RANKL in the presence or absence of IFN-λ1 (100 ng/ml) for 72 h. g Relative mRNA expression of mitf, Ctsk, CTR, and OC-STAMP during treatment with LPS in the presence or absence of IFN-λ1 (100 ng/ml) for 72 h. h Relative mRNA expression of IL-1β, IL-6, and TNF-α during LPS-induced osteoclastogenesis in the presence or absence of IFN-λ1 (100 ng/ml). The data in the figures represent the averages ± SD. Significant differences are indicated as * p

Techniques Used: Translocation Assay, Expressing, Immunofluorescence, Staining, Confocal Microscopy, Software

16) Product Images from "Attenuated RANKL-induced cytotoxicity by Portulaca oleracea ethanol extract enhances RANKL-mediated osteoclastogenesis"

Article Title: Attenuated RANKL-induced cytotoxicity by Portulaca oleracea ethanol extract enhances RANKL-mediated osteoclastogenesis

Journal: BMC Complementary and Alternative Medicine

doi: 10.1186/s12906-015-0770-9

Effects of Portulaca oleracea ethanol extract (POEE) on receptor activator of NF-κB ligand (RANKL)-induced free cytosolic Ca 2+ ([Ca 2+ ] i ) oscillations and nuclear factor of activated T-cell c1 (NFATc1) amplification. a Isolated bone marrow-derived macrophages (BMMs) were plated on cover glass and cultured for 48 h in the presence of RANKL (50 ng/mL). After incubation, [Ca 2+ ] i was measured using Fura-2 AM fluorescent dye as described in “ Materials and Methods .” To confirm the generation of RANKL-induced [Ca 2+ ] i oscillations, cells were initially perfused with regular HEPES buffer, and then acutely treated with 25, 50, and 100 μg/mL of POEE diluted in regular HEPES buffer for the indicated time. Each trace presents the [Ca 2+ ] i response of a single cell. b Isolated BMMs were treated with RANKL (50 ng/mL) for the indicated time with or without POEE (50 μg/mL). Following incubation, whole proteins were collected and used for determining NFATc1 expression. β-actin was used for loading control. NFATc1 expression is shown as the mean of the ratio (NFATc1/β-actin)
Figure Legend Snippet: Effects of Portulaca oleracea ethanol extract (POEE) on receptor activator of NF-κB ligand (RANKL)-induced free cytosolic Ca 2+ ([Ca 2+ ] i ) oscillations and nuclear factor of activated T-cell c1 (NFATc1) amplification. a Isolated bone marrow-derived macrophages (BMMs) were plated on cover glass and cultured for 48 h in the presence of RANKL (50 ng/mL). After incubation, [Ca 2+ ] i was measured using Fura-2 AM fluorescent dye as described in “ Materials and Methods .” To confirm the generation of RANKL-induced [Ca 2+ ] i oscillations, cells were initially perfused with regular HEPES buffer, and then acutely treated with 25, 50, and 100 μg/mL of POEE diluted in regular HEPES buffer for the indicated time. Each trace presents the [Ca 2+ ] i response of a single cell. b Isolated BMMs were treated with RANKL (50 ng/mL) for the indicated time with or without POEE (50 μg/mL). Following incubation, whole proteins were collected and used for determining NFATc1 expression. β-actin was used for loading control. NFATc1 expression is shown as the mean of the ratio (NFATc1/β-actin)

Techniques Used: Amplification, Isolation, Derivative Assay, Cell Culture, Incubation, Expressing

17) Product Images from "Dual roles of QOA-8a in antiosteoporosis: a combination of bone anabolic and anti-resorptive effects"

Article Title: Dual roles of QOA-8a in antiosteoporosis: a combination of bone anabolic and anti-resorptive effects

Journal: Acta Pharmacologica Sinica

doi: 10.1038/aps.2017.63

Signaling involved in inhibiting osteoclast formation induced by QOA-8a. (A) Osteoclast precursors (RAW264.7 cells) were pretreated with vehicle (DMSO) or with 5 μmol/L QOA-8a and stimulated with RANKL in a time-course experiment, and Western blot analysis of ERK1/2, JNK, p38, p65, IκBα activating phosphorylation. (B) RAW264.7 cell cultures incubated on vehicle or with QOA-8a in the presence of RANKL for 1 or 2 d. Western blot analysis of c-Fos and NFATc1 proteins, normalized versus actin. (C) Real-time qPCR of c-Fos and NFATc1 expression, normalized with actin expression. Mean±SD. n =3. ** P
Figure Legend Snippet: Signaling involved in inhibiting osteoclast formation induced by QOA-8a. (A) Osteoclast precursors (RAW264.7 cells) were pretreated with vehicle (DMSO) or with 5 μmol/L QOA-8a and stimulated with RANKL in a time-course experiment, and Western blot analysis of ERK1/2, JNK, p38, p65, IκBα activating phosphorylation. (B) RAW264.7 cell cultures incubated on vehicle or with QOA-8a in the presence of RANKL for 1 or 2 d. Western blot analysis of c-Fos and NFATc1 proteins, normalized versus actin. (C) Real-time qPCR of c-Fos and NFATc1 expression, normalized with actin expression. Mean±SD. n =3. ** P

Techniques Used: Western Blot, Incubation, Real-time Polymerase Chain Reaction, Expressing

18) Product Images from "Cordycepin Accelerates Osteoblast Mineralization and Attenuates Osteoclast Differentiation In Vitro"

Article Title: Cordycepin Accelerates Osteoblast Mineralization and Attenuates Osteoclast Differentiation In Vitro

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

doi: 10.1155/2018/5892957

Cordycepin inhibits RANKL-mediated induction of marker expression in osteoclastogenesis. RAW264.7 cells treated with 25 ng/ml RANKL with or without cordycepin (0.1, 0.5, and 1 μ M) for 72 h. (a) Protein expressions of TRAF6, cFOS, phospho-cFOS, and NFATc1 were analyzed by western blot analysis. (b) Relative protein expression levels compared to β -actin were analyzed by image J. OSCAR (c), cathepsin K (d), and MMP9 (e) relative mRNA expression levels were analyzed by real-time RT-PCR.
Figure Legend Snippet: Cordycepin inhibits RANKL-mediated induction of marker expression in osteoclastogenesis. RAW264.7 cells treated with 25 ng/ml RANKL with or without cordycepin (0.1, 0.5, and 1 μ M) for 72 h. (a) Protein expressions of TRAF6, cFOS, phospho-cFOS, and NFATc1 were analyzed by western blot analysis. (b) Relative protein expression levels compared to β -actin were analyzed by image J. OSCAR (c), cathepsin K (d), and MMP9 (e) relative mRNA expression levels were analyzed by real-time RT-PCR.

Techniques Used: Marker, Expressing, Western Blot, Quantitative RT-PCR

19) Product Images from "Curcumin has immunomodulatory effects on RANKL‐stimulated osteoclastogenesis in vitro and titanium nanoparticle‐induced bone loss in vivo. Curcumin has immunomodulatory effects on RANKL‐stimulated osteoclastogenesis in vitro and titanium nanoparticle‐induced bone loss in vivo"

Article Title: Curcumin has immunomodulatory effects on RANKL‐stimulated osteoclastogenesis in vitro and titanium nanoparticle‐induced bone loss in vivo. Curcumin has immunomodulatory effects on RANKL‐stimulated osteoclastogenesis in vitro and titanium nanoparticle‐induced bone loss in vivo

Journal: Journal of Cellular and Molecular Medicine

doi: 10.1111/jcmm.14842

Curcumin ameliorated the activation of Akt and NF‐κB p65 phosphorylation but had no inhibitory effect on the MAPK pathways. (A and B), RAW264.7 cells were pretreated with or without curcumin for 4 h and then with 100 ng/mL RANKL for indicated time periods (0, 5, 15 or 30 min). Then, the cells were collected and lysed for Western blot analysis. C, The relative grey levels corresponding to p‐ERK, p‐JNK and p‐p38 were quantified and were normalized to β‐actin using ImageJ software. D, The relative grey levels corresponding to p‐Akt, p‐p65 and p‐IκBα were quantified and normalized to β‐actin using ImageJ software. E, RAW264.7 cells were pretreated with or without 20 μmol/L curcumin for 4 h and then stimulated by RANKL for 30 min. The cells were prepared for immunofluorescence staining of p65. F, RAW264.7 cells were cultured in osteoclast induction medium with or without 20 μmol/L curcumin for 1, 3 or 5 d. Cells were then collected and lysed for Western blot analysis. G, The relative grey levels corresponding to c‐fos and NFATc1 were quantified and normalized to β‐actin using ImageJ software. Data are presented as mean ± SD; * P
Figure Legend Snippet: Curcumin ameliorated the activation of Akt and NF‐κB p65 phosphorylation but had no inhibitory effect on the MAPK pathways. (A and B), RAW264.7 cells were pretreated with or without curcumin for 4 h and then with 100 ng/mL RANKL for indicated time periods (0, 5, 15 or 30 min). Then, the cells were collected and lysed for Western blot analysis. C, The relative grey levels corresponding to p‐ERK, p‐JNK and p‐p38 were quantified and were normalized to β‐actin using ImageJ software. D, The relative grey levels corresponding to p‐Akt, p‐p65 and p‐IκBα were quantified and normalized to β‐actin using ImageJ software. E, RAW264.7 cells were pretreated with or without 20 μmol/L curcumin for 4 h and then stimulated by RANKL for 30 min. The cells were prepared for immunofluorescence staining of p65. F, RAW264.7 cells were cultured in osteoclast induction medium with or without 20 μmol/L curcumin for 1, 3 or 5 d. Cells were then collected and lysed for Western blot analysis. G, The relative grey levels corresponding to c‐fos and NFATc1 were quantified and normalized to β‐actin using ImageJ software. Data are presented as mean ± SD; * P

Techniques Used: Activation Assay, Western Blot, Software, Immunofluorescence, Staining, Cell Culture

Schematic illustration of curcumin has immunomodulatory and inhibitory effects on RANKL‐induced osteoclast formation. Curcumin attenuated the up‐regulation of Akt and NF‐κB p65 phosphorylation and the activation of the downstream transcription factor NFATc1. In addition, curcumin created an immunomodulatory microenvironment and promoted macrophage polarization from the M1‐type to the M2‐type phenotype
Figure Legend Snippet: Schematic illustration of curcumin has immunomodulatory and inhibitory effects on RANKL‐induced osteoclast formation. Curcumin attenuated the up‐regulation of Akt and NF‐κB p65 phosphorylation and the activation of the downstream transcription factor NFATc1. In addition, curcumin created an immunomodulatory microenvironment and promoted macrophage polarization from the M1‐type to the M2‐type phenotype

Techniques Used: Activation Assay

Curcumin down‐regulated the expression of RANKL‐induced osteoclast‐related genes including NFATc1, c‐fos, Oscar and Sema‐4A in vitro. A, BMMs were cultured and induced in osteoclast induction medium containing various curcumin concentrations (0, 1.25, 5 or 20 μmol/L) for 5 d. B, BMMs were incubated in osteoclast induction medium with or without 20 μmol/L curcumin for 1, 3 or 5 d. The expression of osteoclast‐related genes was quantified by RT‐PCR. Data are presented as mean ± SD; * P
Figure Legend Snippet: Curcumin down‐regulated the expression of RANKL‐induced osteoclast‐related genes including NFATc1, c‐fos, Oscar and Sema‐4A in vitro. A, BMMs were cultured and induced in osteoclast induction medium containing various curcumin concentrations (0, 1.25, 5 or 20 μmol/L) for 5 d. B, BMMs were incubated in osteoclast induction medium with or without 20 μmol/L curcumin for 1, 3 or 5 d. The expression of osteoclast‐related genes was quantified by RT‐PCR. Data are presented as mean ± SD; * P

Techniques Used: Expressing, In Vitro, Cell Culture, Incubation, Reverse Transcription Polymerase Chain Reaction

20) Product Images from "TRAIL inhibits RANK signaling and suppresses osteoclast activation via inhibiting lipid raft assembly and TRAF6 recruitment"

Article Title: TRAIL inhibits RANK signaling and suppresses osteoclast activation via inhibiting lipid raft assembly and TRAF6 recruitment

Journal: Cell Death & Disease

doi: 10.1038/s41419-019-1353-3

TRAIL inhibits RANKL-induced osteoclast differentiation and activation of NFATc1. a Bone marrow-derived macrophages (BMMs) from wild type (WT) and TRAIL-R knockout ( Trail-r −/− ) mice were plated in 96-well plates and stimulated with the RANKL (50 ng/ml) + M-CSF (20 ng/ml), TRAIL (500 ng/ml), or RANKL + M-CSF + TRAIL as indicated in the figure. After 10 days, cells were analyzed for osteoclast differentiation. After incubation, cells were subjected to a tartrate-resistant acid phosphatase (TRAP) assay. Cell morphology was examined by light microscopy (Scale bars, 100 µm), and the number of TRAP-positive multinuclear cells was quantified in ( b ). ** p
Figure Legend Snippet: TRAIL inhibits RANKL-induced osteoclast differentiation and activation of NFATc1. a Bone marrow-derived macrophages (BMMs) from wild type (WT) and TRAIL-R knockout ( Trail-r −/− ) mice were plated in 96-well plates and stimulated with the RANKL (50 ng/ml) + M-CSF (20 ng/ml), TRAIL (500 ng/ml), or RANKL + M-CSF + TRAIL as indicated in the figure. After 10 days, cells were analyzed for osteoclast differentiation. After incubation, cells were subjected to a tartrate-resistant acid phosphatase (TRAP) assay. Cell morphology was examined by light microscopy (Scale bars, 100 µm), and the number of TRAP-positive multinuclear cells was quantified in ( b ). ** p

Techniques Used: Activation Assay, Derivative Assay, Knock-Out, Mouse Assay, Incubation, TRAP Assay, Light Microscopy

21) Product Images from "Synthetic FXR Agonist GW4064 Is a Modulator of Multiple G Protein–Coupled Receptors"

Article Title: Synthetic FXR Agonist GW4064 Is a Modulator of Multiple G Protein–Coupled Receptors

Journal: Molecular Endocrinology

doi: 10.1210/me.2013-1353

GW4064 induces NFAT translocation and calcineurin activation by a PI-PLC–dependent mechanism. A, GW4064 (GW) induction of intracellular Ca 2+ ) in a FACSCalibur flow cytometer. Data are representative of 3 independent experiments showing identical patterns. FITC, fluorescein isothiocyanate; V, vehicle. B, HEK cells in 10-cm dishes were treated with 1 μM GW4064, ionomycin (1 μM), or FK506 (10 μM) for 30 minutes and then were assayed for calcineurin activity using a colorimetric calcineurin assay kit. Recombinant human calcineurin (CaN, 40 U) was used as a positive control. Data represent nanomoles of PO 4 released (values are normalized to control) and are means ± SEM from 3 representative experiments performed in triplicate. C, HEK cells in chamber slides were treated with 1 μM GW4064 or ionomycin (I, 1 μM) for 30 minutes, and endogenous NFATc1 was detected by immunocytochemical analysis. The nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Magnification, ×40; bars, 50 μm. Data are representative of 3 independent experiments. D, HEK cells in 24-well plates were transfected with 200 ng of NFAT-RE-Luc, and 100 ng of EGFPC1. Cells were then treated with the indicated compounds for 30 minutes followed by treatment with 1 μM GW4064 for 24 hours. Normalized luciferase values were then plotted as percent inhibition of GW4064 response. Data are means ± SEM of 3 independent experiments performed in duplicate. *, P
Figure Legend Snippet: GW4064 induces NFAT translocation and calcineurin activation by a PI-PLC–dependent mechanism. A, GW4064 (GW) induction of intracellular Ca 2+ ) in a FACSCalibur flow cytometer. Data are representative of 3 independent experiments showing identical patterns. FITC, fluorescein isothiocyanate; V, vehicle. B, HEK cells in 10-cm dishes were treated with 1 μM GW4064, ionomycin (1 μM), or FK506 (10 μM) for 30 minutes and then were assayed for calcineurin activity using a colorimetric calcineurin assay kit. Recombinant human calcineurin (CaN, 40 U) was used as a positive control. Data represent nanomoles of PO 4 released (values are normalized to control) and are means ± SEM from 3 representative experiments performed in triplicate. C, HEK cells in chamber slides were treated with 1 μM GW4064 or ionomycin (I, 1 μM) for 30 minutes, and endogenous NFATc1 was detected by immunocytochemical analysis. The nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Magnification, ×40; bars, 50 μm. Data are representative of 3 independent experiments. D, HEK cells in 24-well plates were transfected with 200 ng of NFAT-RE-Luc, and 100 ng of EGFPC1. Cells were then treated with the indicated compounds for 30 minutes followed by treatment with 1 μM GW4064 for 24 hours. Normalized luciferase values were then plotted as percent inhibition of GW4064 response. Data are means ± SEM of 3 independent experiments performed in duplicate. *, P

Techniques Used: Translocation Assay, Activation Assay, Planar Chromatography, Flow Cytometry, Cytometry, Activity Assay, Recombinant, Positive Control, Staining, Transfection, Luciferase, Inhibition

22) Product Images from "Curcumin has immunomodulatory effects on RANKL‐stimulated osteoclastogenesis in vitro and titanium nanoparticle‐induced bone loss in vivo. Curcumin has immunomodulatory effects on RANKL‐stimulated osteoclastogenesis in vitro and titanium nanoparticle‐induced bone loss in vivo"

Article Title: Curcumin has immunomodulatory effects on RANKL‐stimulated osteoclastogenesis in vitro and titanium nanoparticle‐induced bone loss in vivo. Curcumin has immunomodulatory effects on RANKL‐stimulated osteoclastogenesis in vitro and titanium nanoparticle‐induced bone loss in vivo

Journal: Journal of Cellular and Molecular Medicine

doi: 10.1111/jcmm.14842

Curcumin ameliorated the activation of Akt and NF‐κB p65 phosphorylation but had no inhibitory effect on the MAPK pathways. (A and B), RAW264.7 cells were pretreated with or without curcumin for 4 h and then with 100 ng/mL RANKL for indicated time periods (0, 5, 15 or 30 min). Then, the cells were collected and lysed for Western blot analysis. C, The relative grey levels corresponding to p‐ERK, p‐JNK and p‐p38 were quantified and were normalized to β‐actin using ImageJ software. D, The relative grey levels corresponding to p‐Akt, p‐p65 and p‐IκBα were quantified and normalized to β‐actin using ImageJ software. E, RAW264.7 cells were pretreated with or without 20 μmol/L curcumin for 4 h and then stimulated by RANKL for 30 min. The cells were prepared for immunofluorescence staining of p65. F, RAW264.7 cells were cultured in osteoclast induction medium with or without 20 μmol/L curcumin for 1, 3 or 5 d. Cells were then collected and lysed for Western blot analysis. G, The relative grey levels corresponding to c‐fos and NFATc1 were quantified and normalized to β‐actin using ImageJ software. Data are presented as mean ± SD; * P
Figure Legend Snippet: Curcumin ameliorated the activation of Akt and NF‐κB p65 phosphorylation but had no inhibitory effect on the MAPK pathways. (A and B), RAW264.7 cells were pretreated with or without curcumin for 4 h and then with 100 ng/mL RANKL for indicated time periods (0, 5, 15 or 30 min). Then, the cells were collected and lysed for Western blot analysis. C, The relative grey levels corresponding to p‐ERK, p‐JNK and p‐p38 were quantified and were normalized to β‐actin using ImageJ software. D, The relative grey levels corresponding to p‐Akt, p‐p65 and p‐IκBα were quantified and normalized to β‐actin using ImageJ software. E, RAW264.7 cells were pretreated with or without 20 μmol/L curcumin for 4 h and then stimulated by RANKL for 30 min. The cells were prepared for immunofluorescence staining of p65. F, RAW264.7 cells were cultured in osteoclast induction medium with or without 20 μmol/L curcumin for 1, 3 or 5 d. Cells were then collected and lysed for Western blot analysis. G, The relative grey levels corresponding to c‐fos and NFATc1 were quantified and normalized to β‐actin using ImageJ software. Data are presented as mean ± SD; * P

Techniques Used: Activation Assay, Western Blot, Software, Immunofluorescence, Staining, Cell Culture

Schematic illustration of curcumin has immunomodulatory and inhibitory effects on RANKL‐induced osteoclast formation. Curcumin attenuated the up‐regulation of Akt and NF‐κB p65 phosphorylation and the activation of the downstream transcription factor NFATc1. In addition, curcumin created an immunomodulatory microenvironment and promoted macrophage polarization from the M1‐type to the M2‐type phenotype
Figure Legend Snippet: Schematic illustration of curcumin has immunomodulatory and inhibitory effects on RANKL‐induced osteoclast formation. Curcumin attenuated the up‐regulation of Akt and NF‐κB p65 phosphorylation and the activation of the downstream transcription factor NFATc1. In addition, curcumin created an immunomodulatory microenvironment and promoted macrophage polarization from the M1‐type to the M2‐type phenotype

Techniques Used: Activation Assay

Curcumin down‐regulated the expression of RANKL‐induced osteoclast‐related genes including NFATc1, c‐fos, Oscar and Sema‐4A in vitro. A, BMMs were cultured and induced in osteoclast induction medium containing various curcumin concentrations (0, 1.25, 5 or 20 μmol/L) for 5 d. B, BMMs were incubated in osteoclast induction medium with or without 20 μmol/L curcumin for 1, 3 or 5 d. The expression of osteoclast‐related genes was quantified by RT‐PCR. Data are presented as mean ± SD; * P
Figure Legend Snippet: Curcumin down‐regulated the expression of RANKL‐induced osteoclast‐related genes including NFATc1, c‐fos, Oscar and Sema‐4A in vitro. A, BMMs were cultured and induced in osteoclast induction medium containing various curcumin concentrations (0, 1.25, 5 or 20 μmol/L) for 5 d. B, BMMs were incubated in osteoclast induction medium with or without 20 μmol/L curcumin for 1, 3 or 5 d. The expression of osteoclast‐related genes was quantified by RT‐PCR. Data are presented as mean ± SD; * P

Techniques Used: Expressing, In Vitro, Cell Culture, Incubation, Reverse Transcription Polymerase Chain Reaction

23) Product Images from "Abnormal inhibition of osteoclastogenesis by mesenchymal stem cells through the miR-4284/CXCL5 axis in ankylosing spondylitis"

Article Title: Abnormal inhibition of osteoclastogenesis by mesenchymal stem cells through the miR-4284/CXCL5 axis in ankylosing spondylitis

Journal: Cell Death & Disease

doi: 10.1038/s41419-019-1448-x

Inhibition of osteoclastogenesis by ASMSCs compared with HDMSCs. CD14 + monocytes were cultured with HDMSCs or ASMSCs in the presence of M-CSF and RANKL. a Representative images of TRAP staining of osteoclasts co-cultured at different time points (× 100). b The number of TRAP + osteoclasts in each well from cultures at different time points is shown. c Representative images of osteoclasts stained with FITC-phalloidin at different time points (× 200). d Representative images for bone resorption assays at different time points ( × 200). Cells cultured with HDMSCs or ASMSCs on bovine cortical slides were stained with toluidine blue. e Pit formation on each slide was assessed. f mRNA expression levels of TRAP, CTSK, and NFATc1 in osteoclasts were determined by qPCR on day 9. g Protein levels of TRAP, CTSK, and NFATc1 in osteoclasts were determined by western blot analyses on day 9. h Quantitative data of TRAP, CTSK, and NFATc1 protein levels determined by western blot analyses are shown. i Activation of signaling pathways involved in osteoclastogenesis was determined by western blot analyses on day 9. j Quantitative data for activation of signaling pathways determined by western blot analyses are shown. Values are the mean ± SD of 30 samples per group. The results represent three independent experiments. *, p
Figure Legend Snippet: Inhibition of osteoclastogenesis by ASMSCs compared with HDMSCs. CD14 + monocytes were cultured with HDMSCs or ASMSCs in the presence of M-CSF and RANKL. a Representative images of TRAP staining of osteoclasts co-cultured at different time points (× 100). b The number of TRAP + osteoclasts in each well from cultures at different time points is shown. c Representative images of osteoclasts stained with FITC-phalloidin at different time points (× 200). d Representative images for bone resorption assays at different time points ( × 200). Cells cultured with HDMSCs or ASMSCs on bovine cortical slides were stained with toluidine blue. e Pit formation on each slide was assessed. f mRNA expression levels of TRAP, CTSK, and NFATc1 in osteoclasts were determined by qPCR on day 9. g Protein levels of TRAP, CTSK, and NFATc1 in osteoclasts were determined by western blot analyses on day 9. h Quantitative data of TRAP, CTSK, and NFATc1 protein levels determined by western blot analyses are shown. i Activation of signaling pathways involved in osteoclastogenesis was determined by western blot analyses on day 9. j Quantitative data for activation of signaling pathways determined by western blot analyses are shown. Values are the mean ± SD of 30 samples per group. The results represent three independent experiments. *, p

Techniques Used: Inhibition, Cell Culture, Staining, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Activation Assay

Effect of exogenous CXCL5 on osteoclastogenesis. a Representative images of TRAP staining (× 100), F-actin and bone resorption assays (× 200) of osteoclasts treated with the indicated doses of CXCL5 under osteoclastogenic conditions. b The number of TRAP + osteoclasts in each well on day 9 is shown. c Pit formation on each slide on day 15 was assessed. d mRNA expression levels of TRAP, CTSK, and NFATc1 were determined by qRT-PCR on day 9. e Protein levels of TRAP, CTSK, and NFATc1 were determined by western blot analysis on day 9. f Quantitative data for TRAP, CTSK, and NFATc1 protein levels determined by western blot analyses are shown. g Activation of signaling pathways involved in osteoclastogenesis was determined by western blot analyses on day 9. h Quantitative data for activation of signaling pathways determined by western blot analyses are shown. Data are presented as the mean ± SD ( n = 18). The results represent three independent experiments. *, p
Figure Legend Snippet: Effect of exogenous CXCL5 on osteoclastogenesis. a Representative images of TRAP staining (× 100), F-actin and bone resorption assays (× 200) of osteoclasts treated with the indicated doses of CXCL5 under osteoclastogenic conditions. b The number of TRAP + osteoclasts in each well on day 9 is shown. c Pit formation on each slide on day 15 was assessed. d mRNA expression levels of TRAP, CTSK, and NFATc1 were determined by qRT-PCR on day 9. e Protein levels of TRAP, CTSK, and NFATc1 were determined by western blot analysis on day 9. f Quantitative data for TRAP, CTSK, and NFATc1 protein levels determined by western blot analyses are shown. g Activation of signaling pathways involved in osteoclastogenesis was determined by western blot analyses on day 9. h Quantitative data for activation of signaling pathways determined by western blot analyses are shown. Data are presented as the mean ± SD ( n = 18). The results represent three independent experiments. *, p

Techniques Used: Staining, Expressing, Quantitative RT-PCR, Western Blot, Activation Assay

24) Product Images from "Epothilone B prevents lipopolysaccharide-induced inflammatory osteolysis through suppressing osteoclastogenesis via STAT3 signaling pathway"

Article Title: Epothilone B prevents lipopolysaccharide-induced inflammatory osteolysis through suppressing osteoclastogenesis via STAT3 signaling pathway

Journal: Aging (Albany NY)

doi: 10.18632/aging.103337

Epothilone B suppressed NFATc1 nuclear translocation and the expression of marker genes during osteoclastogenesis. ( F ) Relative expression of marker genes in the procedure of RANKL or LPS induced osteoclastogenesis on protein level. ( G ) Quantification of CTSK, MMP9, c-Fos, NFATc1 and CD9 relative to β-actin. Data in the figures represent mean ± SD. N.S. represented no significant difference. *p
Figure Legend Snippet: Epothilone B suppressed NFATc1 nuclear translocation and the expression of marker genes during osteoclastogenesis. ( F ) Relative expression of marker genes in the procedure of RANKL or LPS induced osteoclastogenesis on protein level. ( G ) Quantification of CTSK, MMP9, c-Fos, NFATc1 and CD9 relative to β-actin. Data in the figures represent mean ± SD. N.S. represented no significant difference. *p

Techniques Used: Translocation Assay, Expressing, Marker

Epothilone B suppressed NFATc1 nuclear translocation and the expression of marker genes during osteoclastogenesis. ( A ) Representative images of immunofluorescence staining of the nuclear translocation of NFATc1 in the absence of presence of Epothilone B. Scale bar = 800 μm. ( B ) Quantitative analysis of the percentage of positive cells (NFATc1 translocation from cytosol to nuclear) in all cells. ( C ) Quantitative analysis of the mean intensity of NFATc1 in the cells nuclear. ( D ) Relative expression of marker genes in the procedure of osteoclastogenesis from monocytes to mature osteoclasts on mRNA level. ( E ) Relative expression of marker genes in the early stage of osteoclastogenesis on mRNA level.
Figure Legend Snippet: Epothilone B suppressed NFATc1 nuclear translocation and the expression of marker genes during osteoclastogenesis. ( A ) Representative images of immunofluorescence staining of the nuclear translocation of NFATc1 in the absence of presence of Epothilone B. Scale bar = 800 μm. ( B ) Quantitative analysis of the percentage of positive cells (NFATc1 translocation from cytosol to nuclear) in all cells. ( C ) Quantitative analysis of the mean intensity of NFATc1 in the cells nuclear. ( D ) Relative expression of marker genes in the procedure of osteoclastogenesis from monocytes to mature osteoclasts on mRNA level. ( E ) Relative expression of marker genes in the early stage of osteoclastogenesis on mRNA level.

Techniques Used: Translocation Assay, Expressing, Marker, Immunofluorescence, Staining

25) Product Images from "Bruton's tyrosine kinase (Btk) inhibitor tirabrutinib suppresses osteoclastic bone resorption"

Article Title: Bruton's tyrosine kinase (Btk) inhibitor tirabrutinib suppresses osteoclastic bone resorption

Journal: Bone Reports

doi: 10.1016/j.bonr.2019.100201

Btk mediates Osteoclast signaling pathway. (a, b) Primary human osteoclast precursors were stimulated with M-CSF and RANKL for 48 h. Intracellular phosphorylated Btk (pBtk), total Btk protein (Btk), phosphorylated Lyn (pLyn), total Lyn protein (Lyn), phosphorylated Gab2 (pGab2), total Gab2 protein (Gab2), phosphorylated PLCγ2 (pPLCγ2), total PLCγ2 protein (PLCγ2), phosphorylated BLNK, total BLNK and NFATc1 were detected by Western blot analysis. (b) After immunoprecipitation with an anti-Tec antibody, phosphorylated tyrosine (pTyr) and Tec protein (Tec) were detected by Western blot analysis.
Figure Legend Snippet: Btk mediates Osteoclast signaling pathway. (a, b) Primary human osteoclast precursors were stimulated with M-CSF and RANKL for 48 h. Intracellular phosphorylated Btk (pBtk), total Btk protein (Btk), phosphorylated Lyn (pLyn), total Lyn protein (Lyn), phosphorylated Gab2 (pGab2), total Gab2 protein (Gab2), phosphorylated PLCγ2 (pPLCγ2), total PLCγ2 protein (PLCγ2), phosphorylated BLNK, total BLNK and NFATc1 were detected by Western blot analysis. (b) After immunoprecipitation with an anti-Tec antibody, phosphorylated tyrosine (pTyr) and Tec protein (Tec) were detected by Western blot analysis.

Techniques Used: Western Blot, Immunoprecipitation

RANKL-RANK signaling pathway via Btk in osteoclast differentiation Tec family kinases are activated by RANK and bind to scaffolding proteins (BLNK or SLP76) activated by ITAM signaling, thereby forming a complex that cooperates to activate PLCγ2. PLCγ2 in turn stimulates calcium signaling, which is required for activation of NFATc1, a transcription factor essential for osteoclast differentiation. Our data suggest that ITAM signaling may be bypassed by RANK-RANKL signaling via Btk/Tec.
Figure Legend Snippet: RANKL-RANK signaling pathway via Btk in osteoclast differentiation Tec family kinases are activated by RANK and bind to scaffolding proteins (BLNK or SLP76) activated by ITAM signaling, thereby forming a complex that cooperates to activate PLCγ2. PLCγ2 in turn stimulates calcium signaling, which is required for activation of NFATc1, a transcription factor essential for osteoclast differentiation. Our data suggest that ITAM signaling may be bypassed by RANK-RANKL signaling via Btk/Tec.

Techniques Used: Scaffolding, Activation Assay

26) Product Images from "Curcumin has immunomodulatory effects on RANKL‐stimulated osteoclastogenesis in vitro and titanium nanoparticle‐induced bone loss in vivo. Curcumin has immunomodulatory effects on RANKL‐stimulated osteoclastogenesis in vitro and titanium nanoparticle‐induced bone loss in vivo"

Article Title: Curcumin has immunomodulatory effects on RANKL‐stimulated osteoclastogenesis in vitro and titanium nanoparticle‐induced bone loss in vivo. Curcumin has immunomodulatory effects on RANKL‐stimulated osteoclastogenesis in vitro and titanium nanoparticle‐induced bone loss in vivo

Journal: Journal of Cellular and Molecular Medicine

doi: 10.1111/jcmm.14842

Curcumin ameliorated the activation of Akt and NF‐κB p65 phosphorylation but had no inhibitory effect on the MAPK pathways. (A and B), RAW264.7 cells were pretreated with or without curcumin for 4 h and then with 100 ng/mL RANKL for indicated time periods (0, 5, 15 or 30 min). Then, the cells were collected and lysed for Western blot analysis. C, The relative grey levels corresponding to p‐ERK, p‐JNK and p‐p38 were quantified and were normalized to β‐actin using ImageJ software. D, The relative grey levels corresponding to p‐Akt, p‐p65 and p‐IκBα were quantified and normalized to β‐actin using ImageJ software. E, RAW264.7 cells were pretreated with or without 20 μmol/L curcumin for 4 h and then stimulated by RANKL for 30 min. The cells were prepared for immunofluorescence staining of p65. F, RAW264.7 cells were cultured in osteoclast induction medium with or without 20 μmol/L curcumin for 1, 3 or 5 d. Cells were then collected and lysed for Western blot analysis. G, The relative grey levels corresponding to c‐fos and NFATc1 were quantified and normalized to β‐actin using ImageJ software. Data are presented as mean ± SD; * P
Figure Legend Snippet: Curcumin ameliorated the activation of Akt and NF‐κB p65 phosphorylation but had no inhibitory effect on the MAPK pathways. (A and B), RAW264.7 cells were pretreated with or without curcumin for 4 h and then with 100 ng/mL RANKL for indicated time periods (0, 5, 15 or 30 min). Then, the cells were collected and lysed for Western blot analysis. C, The relative grey levels corresponding to p‐ERK, p‐JNK and p‐p38 were quantified and were normalized to β‐actin using ImageJ software. D, The relative grey levels corresponding to p‐Akt, p‐p65 and p‐IκBα were quantified and normalized to β‐actin using ImageJ software. E, RAW264.7 cells were pretreated with or without 20 μmol/L curcumin for 4 h and then stimulated by RANKL for 30 min. The cells were prepared for immunofluorescence staining of p65. F, RAW264.7 cells were cultured in osteoclast induction medium with or without 20 μmol/L curcumin for 1, 3 or 5 d. Cells were then collected and lysed for Western blot analysis. G, The relative grey levels corresponding to c‐fos and NFATc1 were quantified and normalized to β‐actin using ImageJ software. Data are presented as mean ± SD; * P

Techniques Used: Activation Assay, Western Blot, Software, Immunofluorescence, Staining, Cell Culture

Schematic illustration of curcumin has immunomodulatory and inhibitory effects on RANKL‐induced osteoclast formation. Curcumin attenuated the up‐regulation of Akt and NF‐κB p65 phosphorylation and the activation of the downstream transcription factor NFATc1. In addition, curcumin created an immunomodulatory microenvironment and promoted macrophage polarization from the M1‐type to the M2‐type phenotype
Figure Legend Snippet: Schematic illustration of curcumin has immunomodulatory and inhibitory effects on RANKL‐induced osteoclast formation. Curcumin attenuated the up‐regulation of Akt and NF‐κB p65 phosphorylation and the activation of the downstream transcription factor NFATc1. In addition, curcumin created an immunomodulatory microenvironment and promoted macrophage polarization from the M1‐type to the M2‐type phenotype

Techniques Used: Activation Assay

Curcumin down‐regulated the expression of RANKL‐induced osteoclast‐related genes including NFATc1, c‐fos, Oscar and Sema‐4A in vitro. A, BMMs were cultured and induced in osteoclast induction medium containing various curcumin concentrations (0, 1.25, 5 or 20 μmol/L) for 5 d. B, BMMs were incubated in osteoclast induction medium with or without 20 μmol/L curcumin for 1, 3 or 5 d. The expression of osteoclast‐related genes was quantified by RT‐PCR. Data are presented as mean ± SD; * P
Figure Legend Snippet: Curcumin down‐regulated the expression of RANKL‐induced osteoclast‐related genes including NFATc1, c‐fos, Oscar and Sema‐4A in vitro. A, BMMs were cultured and induced in osteoclast induction medium containing various curcumin concentrations (0, 1.25, 5 or 20 μmol/L) for 5 d. B, BMMs were incubated in osteoclast induction medium with or without 20 μmol/L curcumin for 1, 3 or 5 d. The expression of osteoclast‐related genes was quantified by RT‐PCR. Data are presented as mean ± SD; * P

Techniques Used: Expressing, In Vitro, Cell Culture, Incubation, Reverse Transcription Polymerase Chain Reaction

27) Product Images from "Titanium particle-mediated osteoclastogenesis may be attenuated via bidirectional ephrin-B2/eph-B4 signaling in vitro"

Article Title: Titanium particle-mediated osteoclastogenesis may be attenuated via bidirectional ephrin-B2/eph-B4 signaling in vitro

Journal: International Journal of Molecular Medicine

doi: 10.3892/ijmm.2018.3780

Schematic diagram demonstrating that ephB4-Fc suppresses osteoclast formation and bone resorption induced by Ti particles. Ti particles activated the NFκB pathway in the presence of RANKL. A total of 3 days later, ephrinB2 expression increased significantly. By adding ephB4-Fc, ephrinB2 was activated, which inhibited the C-FOS/NFATc1 signaling pathway and inhibited the gene expression of cathepsin-K, tartrate-resistant acid phosphatase and matrix metalloproteinase 9, by RANKL and Ti particles. Finally, ephB4-Fc inhibits osteoclasts differentiation and bone resorption by Ti particles. Ti, titanium; ephB4, erythropoietin-producing hepatocellular receptor 4; OC, osteoclast; NF-κB, nuclear factor κB; RANK, receptor activator of NF-κB; RANKL, RANK ligand; TRAP, tartrate-resistant acid phosphatase; C-FOS, Fos proto-oncogene, AP-1 transcription factor subunit; NFATc1, nuclear factor of activated T-cells 1.
Figure Legend Snippet: Schematic diagram demonstrating that ephB4-Fc suppresses osteoclast formation and bone resorption induced by Ti particles. Ti particles activated the NFκB pathway in the presence of RANKL. A total of 3 days later, ephrinB2 expression increased significantly. By adding ephB4-Fc, ephrinB2 was activated, which inhibited the C-FOS/NFATc1 signaling pathway and inhibited the gene expression of cathepsin-K, tartrate-resistant acid phosphatase and matrix metalloproteinase 9, by RANKL and Ti particles. Finally, ephB4-Fc inhibits osteoclasts differentiation and bone resorption by Ti particles. Ti, titanium; ephB4, erythropoietin-producing hepatocellular receptor 4; OC, osteoclast; NF-κB, nuclear factor κB; RANK, receptor activator of NF-κB; RANKL, RANK ligand; TRAP, tartrate-resistant acid phosphatase; C-FOS, Fos proto-oncogene, AP-1 transcription factor subunit; NFATc1, nuclear factor of activated T-cells 1.

Techniques Used: Expressing

(A and B) EphB4-Fc inhibited the NFATc1 signaling pathway caused by Ti and inhibited osteoclast-associated genes and protein expression. The mRNA expression of NFATc1, TRAP, MMP9 and CK was detected by reverse transcription quantitative polymerase chain reaction on days 3 and 5, and the protein expression of NFATc1, C-FOS, CK and TRAP was detected by western blot analysis on day 3. Data are presented as mean ± standard deviation. * P
Figure Legend Snippet: (A and B) EphB4-Fc inhibited the NFATc1 signaling pathway caused by Ti and inhibited osteoclast-associated genes and protein expression. The mRNA expression of NFATc1, TRAP, MMP9 and CK was detected by reverse transcription quantitative polymerase chain reaction on days 3 and 5, and the protein expression of NFATc1, C-FOS, CK and TRAP was detected by western blot analysis on day 3. Data are presented as mean ± standard deviation. * P

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Standard Deviation

28) Product Images from "Involvement of interleukin-23 induced by Porphyromonas endodontalis lipopolysaccharide in osteoclastogenesis"

Article Title: Involvement of interleukin-23 induced by Porphyromonas endodontalis lipopolysaccharide in osteoclastogenesis

Journal: Molecular Medicine Reports

doi: 10.3892/mmr.2016.6041

Conditioned medium from P. endodontalis LPS-treated SH-9 cells stimulates osteoclastogenesis. (A) RAW264.7 cells were treated with conditioned medium from P. endodontalis LPS-treated SH-9 cells, or with unconditioned medium containing the same concentrations of LPS and receptor activator of nuclear factor kB ligand, for 48 h. The cells were fixed and subjected to TRAP staining. Representative fields are presented and TRAP-positive cells are indicated by arrows (scale bar, 50 µm; magnification, ×20). (B) The number of TRAP-positive multinuclear cells with > 3 nuclei were counted. Treatment with conditioned medium increased the levels of TRAP-positive multinuclear cells compared with treatment with control medium. The results are presented as the mean ± standard error. (C) RAW264.7 cells were treated with conditioned medium from P. endodontalis LPS-treated SH-9 cells, or unconditioned medium, for 24 h. Protein expression was detected by western blot analysis. Treatment with conditioned medium increased expression of c-Fos and NFATc1 compared with treatment with control medium. **P
Figure Legend Snippet: Conditioned medium from P. endodontalis LPS-treated SH-9 cells stimulates osteoclastogenesis. (A) RAW264.7 cells were treated with conditioned medium from P. endodontalis LPS-treated SH-9 cells, or with unconditioned medium containing the same concentrations of LPS and receptor activator of nuclear factor kB ligand, for 48 h. The cells were fixed and subjected to TRAP staining. Representative fields are presented and TRAP-positive cells are indicated by arrows (scale bar, 50 µm; magnification, ×20). (B) The number of TRAP-positive multinuclear cells with > 3 nuclei were counted. Treatment with conditioned medium increased the levels of TRAP-positive multinuclear cells compared with treatment with control medium. The results are presented as the mean ± standard error. (C) RAW264.7 cells were treated with conditioned medium from P. endodontalis LPS-treated SH-9 cells, or unconditioned medium, for 24 h. Protein expression was detected by western blot analysis. Treatment with conditioned medium increased expression of c-Fos and NFATc1 compared with treatment with control medium. **P

Techniques Used: Staining, Expressing, Western Blot

Knockdown of IL-23 in P. endodontalis LPS-treated SH-9 cells inhibits osteoclastogenesis. (A) SH-9 cells were transfected with siCont or siIL-23 RNA. The mRNA expression levels of IL-23 were examined by reverse transcription-quantitative polymerase chain reaction. IL-23 mRNA expression levels were reduced by siIL-23, compared with siCont transfection. The results are presented as the mean ± standard error. (B) RAW264.7 cells were treated with conditioned medium from P. endodontalis LPS-treated siCont or siIL-23 cells for 48 h. The cells were fixed and subjected to TRAP staining. Representative fields are presented and TRAP-positive cells are indicated by arrows (scale bar, 50 µm; magnification, ×20). (C) TRAP-positive multinuclear cells with > 3 nuclei were counted. The number of TRAP-positive multinuclear cells was reduced in cells treated with conditioned medium from siIL-23, compared with siCont cells. The results are presented as the mean ± standard error. (D) RAW264.7 cells were treated with conditioned medium from P. endodontalis LPS-treated siCont and siIL-23 cells for 24 h. Protein expression was examined by western blot analysis. Treatment with conditioned medium from siIL-23 cells reduced NFATc1 and c-Fos expression compared with treatment with conditioned medium from siCont cells. **P
Figure Legend Snippet: Knockdown of IL-23 in P. endodontalis LPS-treated SH-9 cells inhibits osteoclastogenesis. (A) SH-9 cells were transfected with siCont or siIL-23 RNA. The mRNA expression levels of IL-23 were examined by reverse transcription-quantitative polymerase chain reaction. IL-23 mRNA expression levels were reduced by siIL-23, compared with siCont transfection. The results are presented as the mean ± standard error. (B) RAW264.7 cells were treated with conditioned medium from P. endodontalis LPS-treated siCont or siIL-23 cells for 48 h. The cells were fixed and subjected to TRAP staining. Representative fields are presented and TRAP-positive cells are indicated by arrows (scale bar, 50 µm; magnification, ×20). (C) TRAP-positive multinuclear cells with > 3 nuclei were counted. The number of TRAP-positive multinuclear cells was reduced in cells treated with conditioned medium from siIL-23, compared with siCont cells. The results are presented as the mean ± standard error. (D) RAW264.7 cells were treated with conditioned medium from P. endodontalis LPS-treated siCont and siIL-23 cells for 24 h. Protein expression was examined by western blot analysis. Treatment with conditioned medium from siIL-23 cells reduced NFATc1 and c-Fos expression compared with treatment with conditioned medium from siCont cells. **P

Techniques Used: Transfection, Expressing, Real-time Polymerase Chain Reaction, Staining, Western Blot

29) Product Images from "(+)-Vitisin A Inhibits Osteoclast Differentiation by Preventing TRAF6 Ubiquitination and TRAF6-TAK1 Formation to Suppress NFATc1 Activation"

Article Title: (+)-Vitisin A Inhibits Osteoclast Differentiation by Preventing TRAF6 Ubiquitination and TRAF6-TAK1 Formation to Suppress NFATc1 Activation

Journal: PLoS ONE

doi: 10.1371/journal.pone.0089159

RANKL-stimulated NFATc1 induction and translocation. The (A) induction and (B) nuclear translocation of NFATc1 by RANKL stimulation were suppressed by (+)-vitisin A (Vt-A). RAW264.7 macrophages were stimulated with RANKL (100 ng/mL) for 48 h in the absence or presence of Vt-A then the total and nuclear protein were extracted as described in methods. Results are expressed as the mean ± SEM for each group from four to five separate experiments normalized for histone (for nuclear protein normalization) or β-actin (for total protein normalization) respectively. *p
Figure Legend Snippet: RANKL-stimulated NFATc1 induction and translocation. The (A) induction and (B) nuclear translocation of NFATc1 by RANKL stimulation were suppressed by (+)-vitisin A (Vt-A). RAW264.7 macrophages were stimulated with RANKL (100 ng/mL) for 48 h in the absence or presence of Vt-A then the total and nuclear protein were extracted as described in methods. Results are expressed as the mean ± SEM for each group from four to five separate experiments normalized for histone (for nuclear protein normalization) or β-actin (for total protein normalization) respectively. *p

Techniques Used: Translocation Assay

TRAF6 is poly-ubiquitinated after RANKL stimulation and RANKL-induced expression of NFATc1 in osteoclast differentiation is dependent on TRAF6. (A) RAW264.7 cells were stimulated with RANKL in the absence or presence of TRAF6 siRNA (the results represented are two separate experiments) or control siRNA. After incubation, cell lyses were immunoprecipitated with anti-TRAF6 antibody. Bound proteins were further immunoblotted with anti-ubiquitin or anti-β-actin as described in methods. (B) RAW264.7 cells were stimulated with RANKL in the presence or absence of TRAF6 siRNA or control siRNA. After stimulation, cells lysates were immunoblotted with anti-NFATc1 or anti-β-actin antibodies. Results are expressed as the mean ± SEM for each group from three to four separate experiments. **p
Figure Legend Snippet: TRAF6 is poly-ubiquitinated after RANKL stimulation and RANKL-induced expression of NFATc1 in osteoclast differentiation is dependent on TRAF6. (A) RAW264.7 cells were stimulated with RANKL in the absence or presence of TRAF6 siRNA (the results represented are two separate experiments) or control siRNA. After incubation, cell lyses were immunoprecipitated with anti-TRAF6 antibody. Bound proteins were further immunoblotted with anti-ubiquitin or anti-β-actin as described in methods. (B) RAW264.7 cells were stimulated with RANKL in the presence or absence of TRAF6 siRNA or control siRNA. After stimulation, cells lysates were immunoblotted with anti-NFATc1 or anti-β-actin antibodies. Results are expressed as the mean ± SEM for each group from three to four separate experiments. **p

Techniques Used: Expressing, Incubation, Immunoprecipitation

Proposed intracellular mechanisms of (+)-vitisin A in suppressing RANKL-induced osteoclastogenesis in RAW264.7 cells. The underlying mechanisms involved interfering with the ubiquitination of TRAF6 and downstream signaling cascades to suppression the expression of osteoclast marker proteins and bone resorption. Furthermore, down-regulation of matrix-degrading enzymes activity (cathepsin K and MMP-9) by (+)-vitisin A might also contribute to the beneficial effect on preventing bone loss. AP-1, activator protein 1; DC-STAMP, dendritic cell-specific transmembrane protein; MAPKs, mitogen-activated protein kinases; MITF, microphthalmia-associated transcription factor; MMP-9, matrix metalloproteinase-9; NFATc1, nuclear factor of activated T cells c1; RANKL, receptor activator of nuclear factor κB (NF-κB) ligand; TAK1, transforming growth factor β-activated kinase (TAK)-1; TRAF6, tumour necrosis factor receptor-associated factor 6.
Figure Legend Snippet: Proposed intracellular mechanisms of (+)-vitisin A in suppressing RANKL-induced osteoclastogenesis in RAW264.7 cells. The underlying mechanisms involved interfering with the ubiquitination of TRAF6 and downstream signaling cascades to suppression the expression of osteoclast marker proteins and bone resorption. Furthermore, down-regulation of matrix-degrading enzymes activity (cathepsin K and MMP-9) by (+)-vitisin A might also contribute to the beneficial effect on preventing bone loss. AP-1, activator protein 1; DC-STAMP, dendritic cell-specific transmembrane protein; MAPKs, mitogen-activated protein kinases; MITF, microphthalmia-associated transcription factor; MMP-9, matrix metalloproteinase-9; NFATc1, nuclear factor of activated T cells c1; RANKL, receptor activator of nuclear factor κB (NF-κB) ligand; TAK1, transforming growth factor β-activated kinase (TAK)-1; TRAF6, tumour necrosis factor receptor-associated factor 6.

Techniques Used: Expressing, Marker, Activity Assay

30) Product Images from "Lycorine suppresses RANKL-induced osteoclastogenesis in vitro and prevents ovariectomy-induced osteoporosis and titanium particle-induced osteolysis in vivo"

Article Title: Lycorine suppresses RANKL-induced osteoclastogenesis in vitro and prevents ovariectomy-induced osteoporosis and titanium particle-induced osteolysis in vivo

Journal: Scientific Reports

doi: 10.1038/srep12853

LY inhibited RANKL-induced osteoclast-specific gene expression in vitro . ( A – H ) Expression of the osteoclast-specific genes TRAP, CTR, CTSK, NFATc1, c-Fos, DC-STAMP, V-ATPase d2, and V-ATPase a3 in BMMs treated with LY (0.4 μM), M -CSF (30 ng/mL) and RANKL (50 ng/mL) for 0, 1, 3 or 5 days. Gene expression was analysed by real-time PCR. RNA expression levels were normalized relative to the expression of GAPDH (* P
Figure Legend Snippet: LY inhibited RANKL-induced osteoclast-specific gene expression in vitro . ( A – H ) Expression of the osteoclast-specific genes TRAP, CTR, CTSK, NFATc1, c-Fos, DC-STAMP, V-ATPase d2, and V-ATPase a3 in BMMs treated with LY (0.4 μM), M -CSF (30 ng/mL) and RANKL (50 ng/mL) for 0, 1, 3 or 5 days. Gene expression was analysed by real-time PCR. RNA expression levels were normalized relative to the expression of GAPDH (* P

Techniques Used: Expressing, In Vitro, Real-time Polymerase Chain Reaction, RNA Expression

LY prevention of OVX-induced bone loss assessed by RT-PCR. ( A – F ) Total RNA was extracted from the left femur and tibia of each mouse and reverse transcribed into cDNA, which was used in RT-PCR as described in the Methods section. The expression levels of TRAP, CTSK, CTR, V-ATPase a3, V-ATPase d2, and NFATc1 were examined (* P
Figure Legend Snippet: LY prevention of OVX-induced bone loss assessed by RT-PCR. ( A – F ) Total RNA was extracted from the left femur and tibia of each mouse and reverse transcribed into cDNA, which was used in RT-PCR as described in the Methods section. The expression levels of TRAP, CTSK, CTR, V-ATPase a3, V-ATPase d2, and NFATc1 were examined (* P

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing

31) Product Images from "Curcumin has immunomodulatory effects on RANKL‐stimulated osteoclastogenesis in vitro and titanium nanoparticle‐induced bone loss in vivo. Curcumin has immunomodulatory effects on RANKL‐stimulated osteoclastogenesis in vitro and titanium nanoparticle‐induced bone loss in vivo"

Article Title: Curcumin has immunomodulatory effects on RANKL‐stimulated osteoclastogenesis in vitro and titanium nanoparticle‐induced bone loss in vivo. Curcumin has immunomodulatory effects on RANKL‐stimulated osteoclastogenesis in vitro and titanium nanoparticle‐induced bone loss in vivo

Journal: Journal of Cellular and Molecular Medicine

doi: 10.1111/jcmm.14842

Curcumin ameliorated the activation of Akt and NF‐κB p65 phosphorylation but had no inhibitory effect on the MAPK pathways. (A and B), RAW264.7 cells were pretreated with or without curcumin for 4 h and then with 100 ng/mL RANKL for indicated time periods (0, 5, 15 or 30 min). Then, the cells were collected and lysed for Western blot analysis. C, The relative grey levels corresponding to p‐ERK, p‐JNK and p‐p38 were quantified and were normalized to β‐actin using ImageJ software. D, The relative grey levels corresponding to p‐Akt, p‐p65 and p‐IκBα were quantified and normalized to β‐actin using ImageJ software. E, RAW264.7 cells were pretreated with or without 20 μmol/L curcumin for 4 h and then stimulated by RANKL for 30 min. The cells were prepared for immunofluorescence staining of p65. F, RAW264.7 cells were cultured in osteoclast induction medium with or without 20 μmol/L curcumin for 1, 3 or 5 d. Cells were then collected and lysed for Western blot analysis. G, The relative grey levels corresponding to c‐fos and NFATc1 were quantified and normalized to β‐actin using ImageJ software. Data are presented as mean ± SD; * P
Figure Legend Snippet: Curcumin ameliorated the activation of Akt and NF‐κB p65 phosphorylation but had no inhibitory effect on the MAPK pathways. (A and B), RAW264.7 cells were pretreated with or without curcumin for 4 h and then with 100 ng/mL RANKL for indicated time periods (0, 5, 15 or 30 min). Then, the cells were collected and lysed for Western blot analysis. C, The relative grey levels corresponding to p‐ERK, p‐JNK and p‐p38 were quantified and were normalized to β‐actin using ImageJ software. D, The relative grey levels corresponding to p‐Akt, p‐p65 and p‐IκBα were quantified and normalized to β‐actin using ImageJ software. E, RAW264.7 cells were pretreated with or without 20 μmol/L curcumin for 4 h and then stimulated by RANKL for 30 min. The cells were prepared for immunofluorescence staining of p65. F, RAW264.7 cells were cultured in osteoclast induction medium with or without 20 μmol/L curcumin for 1, 3 or 5 d. Cells were then collected and lysed for Western blot analysis. G, The relative grey levels corresponding to c‐fos and NFATc1 were quantified and normalized to β‐actin using ImageJ software. Data are presented as mean ± SD; * P

Techniques Used: Activation Assay, Western Blot, Software, Immunofluorescence, Staining, Cell Culture

Schematic illustration of curcumin has immunomodulatory and inhibitory effects on RANKL‐induced osteoclast formation. Curcumin attenuated the up‐regulation of Akt and NF‐κB p65 phosphorylation and the activation of the downstream transcription factor NFATc1. In addition, curcumin created an immunomodulatory microenvironment and promoted macrophage polarization from the M1‐type to the M2‐type phenotype
Figure Legend Snippet: Schematic illustration of curcumin has immunomodulatory and inhibitory effects on RANKL‐induced osteoclast formation. Curcumin attenuated the up‐regulation of Akt and NF‐κB p65 phosphorylation and the activation of the downstream transcription factor NFATc1. In addition, curcumin created an immunomodulatory microenvironment and promoted macrophage polarization from the M1‐type to the M2‐type phenotype

Techniques Used: Activation Assay

Curcumin down‐regulated the expression of RANKL‐induced osteoclast‐related genes including NFATc1, c‐fos, Oscar and Sema‐4A in vitro. A, BMMs were cultured and induced in osteoclast induction medium containing various curcumin concentrations (0, 1.25, 5 or 20 μmol/L) for 5 d. B, BMMs were incubated in osteoclast induction medium with or without 20 μmol/L curcumin for 1, 3 or 5 d. The expression of osteoclast‐related genes was quantified by RT‐PCR. Data are presented as mean ± SD; * P
Figure Legend Snippet: Curcumin down‐regulated the expression of RANKL‐induced osteoclast‐related genes including NFATc1, c‐fos, Oscar and Sema‐4A in vitro. A, BMMs were cultured and induced in osteoclast induction medium containing various curcumin concentrations (0, 1.25, 5 or 20 μmol/L) for 5 d. B, BMMs were incubated in osteoclast induction medium with or without 20 μmol/L curcumin for 1, 3 or 5 d. The expression of osteoclast‐related genes was quantified by RT‐PCR. Data are presented as mean ± SD; * P

Techniques Used: Expressing, In Vitro, Cell Culture, Incubation, Reverse Transcription Polymerase Chain Reaction

32) Product Images from "The Recombinant Protein EphB4-Fc Changes the Ti Particle-Mediated Imbalance of OPG/RANKL via EphrinB2/EphB4 Signaling Pathway and Inhibits the Release of Proinflammatory Factors In Vivo"

Article Title: The Recombinant Protein EphB4-Fc Changes the Ti Particle-Mediated Imbalance of OPG/RANKL via EphrinB2/EphB4 Signaling Pathway and Inhibits the Release of Proinflammatory Factors In Vivo

Journal: Oxidative Medicine and Cellular Longevity

doi: 10.1155/2020/1404915

The expression of ephrinB2 is mediated by c-Fos/NFATc1 signaling pathway. (a) Shows that the ephrinB2 gene is regulated by the c-Fos/NFATc1 signaling pathway through sic-Fos RNA and siNFATc1 RNA. We find that it promotes high expression of ephrinB2 gene and protein with Ti (b, c) and the ephrinB2 protein is located on the surface of the BMMs membrane by immunofluorescence. Data are represented as mean ± standard deviation, n = 3 ( ∗ P
Figure Legend Snippet: The expression of ephrinB2 is mediated by c-Fos/NFATc1 signaling pathway. (a) Shows that the ephrinB2 gene is regulated by the c-Fos/NFATc1 signaling pathway through sic-Fos RNA and siNFATc1 RNA. We find that it promotes high expression of ephrinB2 gene and protein with Ti (b, c) and the ephrinB2 protein is located on the surface of the BMMs membrane by immunofluorescence. Data are represented as mean ± standard deviation, n = 3 ( ∗ P

Techniques Used: Expressing, Immunofluorescence, Standard Deviation

33) Product Images from "Bruton's tyrosine kinase (Btk) inhibitor tirabrutinib suppresses osteoclastic bone resorption"

Article Title: Bruton's tyrosine kinase (Btk) inhibitor tirabrutinib suppresses osteoclastic bone resorption

Journal: Bone Reports

doi: 10.1016/j.bonr.2019.100201

Btk mediates Osteoclast signaling pathway. (a, b) Primary human osteoclast precursors were stimulated with M-CSF and RANKL for 48 h. Intracellular phosphorylated Btk (pBtk), total Btk protein (Btk), phosphorylated Lyn (pLyn), total Lyn protein (Lyn), phosphorylated Gab2 (pGab2), total Gab2 protein (Gab2), phosphorylated PLCγ2 (pPLCγ2), total PLCγ2 protein (PLCγ2), phosphorylated BLNK, total BLNK and NFATc1 were detected by Western blot analysis. (b) After immunoprecipitation with an anti-Tec antibody, phosphorylated tyrosine (pTyr) and Tec protein (Tec) were detected by Western blot analysis.
Figure Legend Snippet: Btk mediates Osteoclast signaling pathway. (a, b) Primary human osteoclast precursors were stimulated with M-CSF and RANKL for 48 h. Intracellular phosphorylated Btk (pBtk), total Btk protein (Btk), phosphorylated Lyn (pLyn), total Lyn protein (Lyn), phosphorylated Gab2 (pGab2), total Gab2 protein (Gab2), phosphorylated PLCγ2 (pPLCγ2), total PLCγ2 protein (PLCγ2), phosphorylated BLNK, total BLNK and NFATc1 were detected by Western blot analysis. (b) After immunoprecipitation with an anti-Tec antibody, phosphorylated tyrosine (pTyr) and Tec protein (Tec) were detected by Western blot analysis.

Techniques Used: Western Blot, Immunoprecipitation

RANKL-RANK signaling pathway via Btk in osteoclast differentiation Tec family kinases are activated by RANK and bind to scaffolding proteins (BLNK or SLP76) activated by ITAM signaling, thereby forming a complex that cooperates to activate PLCγ2. PLCγ2 in turn stimulates calcium signaling, which is required for activation of NFATc1, a transcription factor essential for osteoclast differentiation. Our data suggest that ITAM signaling may be bypassed by RANK-RANKL signaling via Btk/Tec.
Figure Legend Snippet: RANKL-RANK signaling pathway via Btk in osteoclast differentiation Tec family kinases are activated by RANK and bind to scaffolding proteins (BLNK or SLP76) activated by ITAM signaling, thereby forming a complex that cooperates to activate PLCγ2. PLCγ2 in turn stimulates calcium signaling, which is required for activation of NFATc1, a transcription factor essential for osteoclast differentiation. Our data suggest that ITAM signaling may be bypassed by RANK-RANKL signaling via Btk/Tec.

Techniques Used: Scaffolding, Activation Assay

34) Product Images from "Agelasine D Suppresses RANKL-Induced Osteoclastogenesis via Down-Regulation of c-Fos, NFATc1 and NF-κB"

Article Title: Agelasine D Suppresses RANKL-Induced Osteoclastogenesis via Down-Regulation of c-Fos, NFATc1 and NF-κB

Journal: Marine Drugs

doi: 10.3390/md12115643

Schematic diagram of signaling pathways important for RANKL-induced osteoclastogenesis. The inhibitory effect of AD is mediated by blocking the activation of NF-κB and ERK signaling pathways and concomittant down-regulation of c-Fos and NFATc1.
Figure Legend Snippet: Schematic diagram of signaling pathways important for RANKL-induced osteoclastogenesis. The inhibitory effect of AD is mediated by blocking the activation of NF-κB and ERK signaling pathways and concomittant down-regulation of c-Fos and NFATc1.

Techniques Used: Blocking Assay, Activation Assay

BMMs were pretreated with vehicle or indicated concentrations of AD in the presence of M-CSF for 1 h and stimulated with RANKL for indicated times. Total cell lysates were prepared and the expression of c-Fos ( A ) and nuclear factor of activated T cells c1 (NFATc1) ( B ) and the phosphorylation of extracellular signal-related kinase (ERK) ( C ) were analyzed by Western Immunoblot analysis; ( D ) BMMs were treated with vehicle or indicated concentrations of AD in the presence of RANKL and M-CSF for 3 days. Total RNAs were isolated and the mRNA expression of NFATc1 was analyzed by quantitative RT-PCR.
Figure Legend Snippet: BMMs were pretreated with vehicle or indicated concentrations of AD in the presence of M-CSF for 1 h and stimulated with RANKL for indicated times. Total cell lysates were prepared and the expression of c-Fos ( A ) and nuclear factor of activated T cells c1 (NFATc1) ( B ) and the phosphorylation of extracellular signal-related kinase (ERK) ( C ) were analyzed by Western Immunoblot analysis; ( D ) BMMs were treated with vehicle or indicated concentrations of AD in the presence of RANKL and M-CSF for 3 days. Total RNAs were isolated and the mRNA expression of NFATc1 was analyzed by quantitative RT-PCR.

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

35) Product Images from "Cyperenoic acid suppresses osteoclast differentiation and delays bone loss in a senile osteoporosis mouse model by inhibiting non-canonical NF-κB pathway"

Article Title: Cyperenoic acid suppresses osteoclast differentiation and delays bone loss in a senile osteoporosis mouse model by inhibiting non-canonical NF-κB pathway

Journal: Scientific Reports

doi: 10.1038/s41598-018-23912-3

Effect of cyperenoic acid on activation of MAPK, canonical or non-canonical NF-κB pathways and expression of NFATc1 and c-Fos. M-CSF treated BMs were pre-treated with cyperenoic acid (100 μM) or vehicle control DMSO for 30 min and RANKL (100 ng/ml) was used to stimulate cells at the indicated times. Whole cell lysates were analyzed for phosphorylation forms of MAPK and canonical NF-κB ( A ) or non-canonical NF-κB ( B ) by Western blot. ( C – D ) The quantitative band density of phosphorylated p100 and total p52 were normalized by total p100 or β-actin, respectively. ( E ) The level of c-Fos and NFATc1 were analyzed by Western blot. β-actin was used as a loading control. Data were representative of three independent experiments.
Figure Legend Snippet: Effect of cyperenoic acid on activation of MAPK, canonical or non-canonical NF-κB pathways and expression of NFATc1 and c-Fos. M-CSF treated BMs were pre-treated with cyperenoic acid (100 μM) or vehicle control DMSO for 30 min and RANKL (100 ng/ml) was used to stimulate cells at the indicated times. Whole cell lysates were analyzed for phosphorylation forms of MAPK and canonical NF-κB ( A ) or non-canonical NF-κB ( B ) by Western blot. ( C – D ) The quantitative band density of phosphorylated p100 and total p52 were normalized by total p100 or β-actin, respectively. ( E ) The level of c-Fos and NFATc1 were analyzed by Western blot. β-actin was used as a loading control. Data were representative of three independent experiments.

Techniques Used: Activation Assay, Expressing, Western Blot

Effect of cyperenoic acid on expression of osteoclast-related genes. BMs were pre-treated with M-CSF with DMSO and left unstimulated (open bars), M-CSF with DMSO for 30 min, followed by RANKL stimulation (closed bars) or M-CSF with cyperenoic acid (100 µM) for 30 min, followed by RANKL stimulation (hatched bars) for indicated times. Total RNA was extracted and subjected to RT-qPCR. Osteoclast-related genes were nfatc1 ( A ), ctsk ( B ), irf8 ( C ), acp5 ( D ) and cfos ( E ). The data are representative of three independent experiments and presented as the mean ± S.D. *** p
Figure Legend Snippet: Effect of cyperenoic acid on expression of osteoclast-related genes. BMs were pre-treated with M-CSF with DMSO and left unstimulated (open bars), M-CSF with DMSO for 30 min, followed by RANKL stimulation (closed bars) or M-CSF with cyperenoic acid (100 µM) for 30 min, followed by RANKL stimulation (hatched bars) for indicated times. Total RNA was extracted and subjected to RT-qPCR. Osteoclast-related genes were nfatc1 ( A ), ctsk ( B ), irf8 ( C ), acp5 ( D ) and cfos ( E ). The data are representative of three independent experiments and presented as the mean ± S.D. *** p

Techniques Used: Expressing, Quantitative RT-PCR

36) Product Images from "Inhibition of RANKL-stimulated osteoclast differentiation by Schisandra chinensis through down-regulation of NFATc1 and c-fos expression"

Article Title: Inhibition of RANKL-stimulated osteoclast differentiation by Schisandra chinensis through down-regulation of NFATc1 and c-fos expression

Journal: BMC Complementary and Alternative Medicine

doi: 10.1186/s12906-018-2331-5

The effects of SC on NFATc1 and c-fos protein expressions in RANKL-stimulated RAW 264.7 cells. RAW 264.7 cells were cultured in the presence of RANKL with the vehicle or SC. After 7 days, total protein was then isolated and protein expression levels were evaluated by Western blot assay. Data are represented as the means ± S.E.M. of three independent experiments. ## p
Figure Legend Snippet: The effects of SC on NFATc1 and c-fos protein expressions in RANKL-stimulated RAW 264.7 cells. RAW 264.7 cells were cultured in the presence of RANKL with the vehicle or SC. After 7 days, total protein was then isolated and protein expression levels were evaluated by Western blot assay. Data are represented as the means ± S.E.M. of three independent experiments. ## p

Techniques Used: Cell Culture, Isolation, Expressing, Western Blot

37) Product Images from "Bruton's tyrosine kinase (Btk) inhibitor tirabrutinib suppresses osteoclastic bone resorption"

Article Title: Bruton's tyrosine kinase (Btk) inhibitor tirabrutinib suppresses osteoclastic bone resorption

Journal: Bone Reports

doi: 10.1016/j.bonr.2019.100201

Btk mediates Osteoclast signaling pathway. (a, b) Primary human osteoclast precursors were stimulated with M-CSF and RANKL for 48 h. Intracellular phosphorylated Btk (pBtk), total Btk protein (Btk), phosphorylated Lyn (pLyn), total Lyn protein (Lyn), phosphorylated Gab2 (pGab2), total Gab2 protein (Gab2), phosphorylated PLCγ2 (pPLCγ2), total PLCγ2 protein (PLCγ2), phosphorylated BLNK, total BLNK and NFATc1 were detected by Western blot analysis. (b) After immunoprecipitation with an anti-Tec antibody, phosphorylated tyrosine (pTyr) and Tec protein (Tec) were detected by Western blot analysis.
Figure Legend Snippet: Btk mediates Osteoclast signaling pathway. (a, b) Primary human osteoclast precursors were stimulated with M-CSF and RANKL for 48 h. Intracellular phosphorylated Btk (pBtk), total Btk protein (Btk), phosphorylated Lyn (pLyn), total Lyn protein (Lyn), phosphorylated Gab2 (pGab2), total Gab2 protein (Gab2), phosphorylated PLCγ2 (pPLCγ2), total PLCγ2 protein (PLCγ2), phosphorylated BLNK, total BLNK and NFATc1 were detected by Western blot analysis. (b) After immunoprecipitation with an anti-Tec antibody, phosphorylated tyrosine (pTyr) and Tec protein (Tec) were detected by Western blot analysis.

Techniques Used: Western Blot, Immunoprecipitation

RANKL-RANK signaling pathway via Btk in osteoclast differentiation Tec family kinases are activated by RANK and bind to scaffolding proteins (BLNK or SLP76) activated by ITAM signaling, thereby forming a complex that cooperates to activate PLCγ2. PLCγ2 in turn stimulates calcium signaling, which is required for activation of NFATc1, a transcription factor essential for osteoclast differentiation. Our data suggest that ITAM signaling may be bypassed by RANK-RANKL signaling via Btk/Tec.
Figure Legend Snippet: RANKL-RANK signaling pathway via Btk in osteoclast differentiation Tec family kinases are activated by RANK and bind to scaffolding proteins (BLNK or SLP76) activated by ITAM signaling, thereby forming a complex that cooperates to activate PLCγ2. PLCγ2 in turn stimulates calcium signaling, which is required for activation of NFATc1, a transcription factor essential for osteoclast differentiation. Our data suggest that ITAM signaling may be bypassed by RANK-RANKL signaling via Btk/Tec.

Techniques Used: Scaffolding, Activation Assay

38) Product Images from "Curcumin has immunomodulatory effects on RANKL‐stimulated osteoclastogenesis in vitro and titanium nanoparticle‐induced bone loss in vivo. Curcumin has immunomodulatory effects on RANKL‐stimulated osteoclastogenesis in vitro and titanium nanoparticle‐induced bone loss in vivo"

Article Title: Curcumin has immunomodulatory effects on RANKL‐stimulated osteoclastogenesis in vitro and titanium nanoparticle‐induced bone loss in vivo. Curcumin has immunomodulatory effects on RANKL‐stimulated osteoclastogenesis in vitro and titanium nanoparticle‐induced bone loss in vivo

Journal: Journal of Cellular and Molecular Medicine

doi: 10.1111/jcmm.14842

Curcumin ameliorated the activation of Akt and NF‐κB p65 phosphorylation but had no inhibitory effect on the MAPK pathways. (A and B), RAW264.7 cells were pretreated with or without curcumin for 4 h and then with 100 ng/mL RANKL for indicated time periods (0, 5, 15 or 30 min). Then, the cells were collected and lysed for Western blot analysis. C, The relative grey levels corresponding to p‐ERK, p‐JNK and p‐p38 were quantified and were normalized to β‐actin using ImageJ software. D, The relative grey levels corresponding to p‐Akt, p‐p65 and p‐IκBα were quantified and normalized to β‐actin using ImageJ software. E, RAW264.7 cells were pretreated with or without 20 μmol/L curcumin for 4 h and then stimulated by RANKL for 30 min. The cells were prepared for immunofluorescence staining of p65. F, RAW264.7 cells were cultured in osteoclast induction medium with or without 20 μmol/L curcumin for 1, 3 or 5 d. Cells were then collected and lysed for Western blot analysis. G, The relative grey levels corresponding to c‐fos and NFATc1 were quantified and normalized to β‐actin using ImageJ software. Data are presented as mean ± SD; * P
Figure Legend Snippet: Curcumin ameliorated the activation of Akt and NF‐κB p65 phosphorylation but had no inhibitory effect on the MAPK pathways. (A and B), RAW264.7 cells were pretreated with or without curcumin for 4 h and then with 100 ng/mL RANKL for indicated time periods (0, 5, 15 or 30 min). Then, the cells were collected and lysed for Western blot analysis. C, The relative grey levels corresponding to p‐ERK, p‐JNK and p‐p38 were quantified and were normalized to β‐actin using ImageJ software. D, The relative grey levels corresponding to p‐Akt, p‐p65 and p‐IκBα were quantified and normalized to β‐actin using ImageJ software. E, RAW264.7 cells were pretreated with or without 20 μmol/L curcumin for 4 h and then stimulated by RANKL for 30 min. The cells were prepared for immunofluorescence staining of p65. F, RAW264.7 cells were cultured in osteoclast induction medium with or without 20 μmol/L curcumin for 1, 3 or 5 d. Cells were then collected and lysed for Western blot analysis. G, The relative grey levels corresponding to c‐fos and NFATc1 were quantified and normalized to β‐actin using ImageJ software. Data are presented as mean ± SD; * P

Techniques Used: Activation Assay, Western Blot, Software, Immunofluorescence, Staining, Cell Culture

Schematic illustration of curcumin has immunomodulatory and inhibitory effects on RANKL‐induced osteoclast formation. Curcumin attenuated the up‐regulation of Akt and NF‐κB p65 phosphorylation and the activation of the downstream transcription factor NFATc1. In addition, curcumin created an immunomodulatory microenvironment and promoted macrophage polarization from the M1‐type to the M2‐type phenotype
Figure Legend Snippet: Schematic illustration of curcumin has immunomodulatory and inhibitory effects on RANKL‐induced osteoclast formation. Curcumin attenuated the up‐regulation of Akt and NF‐κB p65 phosphorylation and the activation of the downstream transcription factor NFATc1. In addition, curcumin created an immunomodulatory microenvironment and promoted macrophage polarization from the M1‐type to the M2‐type phenotype

Techniques Used: Activation Assay

Curcumin down‐regulated the expression of RANKL‐induced osteoclast‐related genes including NFATc1, c‐fos, Oscar and Sema‐4A in vitro. A, BMMs were cultured and induced in osteoclast induction medium containing various curcumin concentrations (0, 1.25, 5 or 20 μmol/L) for 5 d. B, BMMs were incubated in osteoclast induction medium with or without 20 μmol/L curcumin for 1, 3 or 5 d. The expression of osteoclast‐related genes was quantified by RT‐PCR. Data are presented as mean ± SD; * P
Figure Legend Snippet: Curcumin down‐regulated the expression of RANKL‐induced osteoclast‐related genes including NFATc1, c‐fos, Oscar and Sema‐4A in vitro. A, BMMs were cultured and induced in osteoclast induction medium containing various curcumin concentrations (0, 1.25, 5 or 20 μmol/L) for 5 d. B, BMMs were incubated in osteoclast induction medium with or without 20 μmol/L curcumin for 1, 3 or 5 d. The expression of osteoclast‐related genes was quantified by RT‐PCR. Data are presented as mean ± SD; * P

Techniques Used: Expressing, In Vitro, Cell Culture, Incubation, Reverse Transcription Polymerase Chain Reaction

39) Product Images from "Curcumin has immunomodulatory effects on RANKL‐stimulated osteoclastogenesis in vitro and titanium nanoparticle‐induced bone loss in vivo. Curcumin has immunomodulatory effects on RANKL‐stimulated osteoclastogenesis in vitro and titanium nanoparticle‐induced bone loss in vivo"

Article Title: Curcumin has immunomodulatory effects on RANKL‐stimulated osteoclastogenesis in vitro and titanium nanoparticle‐induced bone loss in vivo. Curcumin has immunomodulatory effects on RANKL‐stimulated osteoclastogenesis in vitro and titanium nanoparticle‐induced bone loss in vivo

Journal: Journal of Cellular and Molecular Medicine

doi: 10.1111/jcmm.14842

Curcumin ameliorated the activation of Akt and NF‐κB p65 phosphorylation but had no inhibitory effect on the MAPK pathways. (A and B), RAW264.7 cells were pretreated with or without curcumin for 4 h and then with 100 ng/mL RANKL for indicated time periods (0, 5, 15 or 30 min). Then, the cells were collected and lysed for Western blot analysis. C, The relative grey levels corresponding to p‐ERK, p‐JNK and p‐p38 were quantified and were normalized to β‐actin using ImageJ software. D, The relative grey levels corresponding to p‐Akt, p‐p65 and p‐IκBα were quantified and normalized to β‐actin using ImageJ software. E, RAW264.7 cells were pretreated with or without 20 μmol/L curcumin for 4 h and then stimulated by RANKL for 30 min. The cells were prepared for immunofluorescence staining of p65. F, RAW264.7 cells were cultured in osteoclast induction medium with or without 20 μmol/L curcumin for 1, 3 or 5 d. Cells were then collected and lysed for Western blot analysis. G, The relative grey levels corresponding to c‐fos and NFATc1 were quantified and normalized to β‐actin using ImageJ software. Data are presented as mean ± SD; * P
Figure Legend Snippet: Curcumin ameliorated the activation of Akt and NF‐κB p65 phosphorylation but had no inhibitory effect on the MAPK pathways. (A and B), RAW264.7 cells were pretreated with or without curcumin for 4 h and then with 100 ng/mL RANKL for indicated time periods (0, 5, 15 or 30 min). Then, the cells were collected and lysed for Western blot analysis. C, The relative grey levels corresponding to p‐ERK, p‐JNK and p‐p38 were quantified and were normalized to β‐actin using ImageJ software. D, The relative grey levels corresponding to p‐Akt, p‐p65 and p‐IκBα were quantified and normalized to β‐actin using ImageJ software. E, RAW264.7 cells were pretreated with or without 20 μmol/L curcumin for 4 h and then stimulated by RANKL for 30 min. The cells were prepared for immunofluorescence staining of p65. F, RAW264.7 cells were cultured in osteoclast induction medium with or without 20 μmol/L curcumin for 1, 3 or 5 d. Cells were then collected and lysed for Western blot analysis. G, The relative grey levels corresponding to c‐fos and NFATc1 were quantified and normalized to β‐actin using ImageJ software. Data are presented as mean ± SD; * P

Techniques Used: Activation Assay, Western Blot, Software, Immunofluorescence, Staining, Cell Culture

Schematic illustration of curcumin has immunomodulatory and inhibitory effects on RANKL‐induced osteoclast formation. Curcumin attenuated the up‐regulation of Akt and NF‐κB p65 phosphorylation and the activation of the downstream transcription factor NFATc1. In addition, curcumin created an immunomodulatory microenvironment and promoted macrophage polarization from the M1‐type to the M2‐type phenotype
Figure Legend Snippet: Schematic illustration of curcumin has immunomodulatory and inhibitory effects on RANKL‐induced osteoclast formation. Curcumin attenuated the up‐regulation of Akt and NF‐κB p65 phosphorylation and the activation of the downstream transcription factor NFATc1. In addition, curcumin created an immunomodulatory microenvironment and promoted macrophage polarization from the M1‐type to the M2‐type phenotype

Techniques Used: Activation Assay

Curcumin down‐regulated the expression of RANKL‐induced osteoclast‐related genes including NFATc1, c‐fos, Oscar and Sema‐4A in vitro. A, BMMs were cultured and induced in osteoclast induction medium containing various curcumin concentrations (0, 1.25, 5 or 20 μmol/L) for 5 d. B, BMMs were incubated in osteoclast induction medium with or without 20 μmol/L curcumin for 1, 3 or 5 d. The expression of osteoclast‐related genes was quantified by RT‐PCR. Data are presented as mean ± SD; * P
Figure Legend Snippet: Curcumin down‐regulated the expression of RANKL‐induced osteoclast‐related genes including NFATc1, c‐fos, Oscar and Sema‐4A in vitro. A, BMMs were cultured and induced in osteoclast induction medium containing various curcumin concentrations (0, 1.25, 5 or 20 μmol/L) for 5 d. B, BMMs were incubated in osteoclast induction medium with or without 20 μmol/L curcumin for 1, 3 or 5 d. The expression of osteoclast‐related genes was quantified by RT‐PCR. Data are presented as mean ± SD; * P

Techniques Used: Expressing, In Vitro, Cell Culture, Incubation, Reverse Transcription Polymerase Chain Reaction

40) Product Images from "Titanium particle-mediated osteoclastogenesis may be attenuated via bidirectional ephrin-B2/eph-B4 signaling in vitro"

Article Title: Titanium particle-mediated osteoclastogenesis may be attenuated via bidirectional ephrin-B2/eph-B4 signaling in vitro

Journal: International Journal of Molecular Medicine

doi: 10.3892/ijmm.2018.3780

Schematic diagram demonstrating that ephB4-Fc suppresses osteoclast formation and bone resorption induced by Ti particles. Ti particles activated the NFκB pathway in the presence of RANKL. A total of 3 days later, ephrinB2 expression increased significantly. By adding ephB4-Fc, ephrinB2 was activated, which inhibited the C-FOS/NFATc1 signaling pathway and inhibited the gene expression of cathepsin-K, tartrate-resistant acid phosphatase and matrix metalloproteinase 9, by RANKL and Ti particles. Finally, ephB4-Fc inhibits osteoclasts differentiation and bone resorption by Ti particles. Ti, titanium; ephB4, erythropoietin-producing hepatocellular receptor 4; OC, osteoclast; NF-κB, nuclear factor κB; RANK, receptor activator of NF-κB; RANKL, RANK ligand; TRAP, tartrate-resistant acid phosphatase; C-FOS, Fos proto-oncogene, AP-1 transcription factor subunit; NFATc1, nuclear factor of activated T-cells 1.
Figure Legend Snippet: Schematic diagram demonstrating that ephB4-Fc suppresses osteoclast formation and bone resorption induced by Ti particles. Ti particles activated the NFκB pathway in the presence of RANKL. A total of 3 days later, ephrinB2 expression increased significantly. By adding ephB4-Fc, ephrinB2 was activated, which inhibited the C-FOS/NFATc1 signaling pathway and inhibited the gene expression of cathepsin-K, tartrate-resistant acid phosphatase and matrix metalloproteinase 9, by RANKL and Ti particles. Finally, ephB4-Fc inhibits osteoclasts differentiation and bone resorption by Ti particles. Ti, titanium; ephB4, erythropoietin-producing hepatocellular receptor 4; OC, osteoclast; NF-κB, nuclear factor κB; RANK, receptor activator of NF-κB; RANKL, RANK ligand; TRAP, tartrate-resistant acid phosphatase; C-FOS, Fos proto-oncogene, AP-1 transcription factor subunit; NFATc1, nuclear factor of activated T-cells 1.

Techniques Used: Expressing

(A and B) EphB4-Fc inhibited the NFATc1 signaling pathway caused by Ti and inhibited osteoclast-associated genes and protein expression. The mRNA expression of NFATc1, TRAP, MMP9 and CK was detected by reverse transcription quantitative polymerase chain reaction on days 3 and 5, and the protein expression of NFATc1, C-FOS, CK and TRAP was detected by western blot analysis on day 3. Data are presented as mean ± standard deviation. * P
Figure Legend Snippet: (A and B) EphB4-Fc inhibited the NFATc1 signaling pathway caused by Ti and inhibited osteoclast-associated genes and protein expression. The mRNA expression of NFATc1, TRAP, MMP9 and CK was detected by reverse transcription quantitative polymerase chain reaction on days 3 and 5, and the protein expression of NFATc1, C-FOS, CK and TRAP was detected by western blot analysis on day 3. Data are presented as mean ± standard deviation. * P

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Standard Deviation

Related Articles

Nucleic Acid Electrophoresis:

Article Title: Study on the Mechanism of mTOR-Mediated Autophagy during Electroacupuncture Pretreatment against Cerebral Ischemic Injury
Article Snippet: .. A 60 μ g aliquot of proteins from each sample was extracted by applying 10% SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane, which was then incubated with the specific antibodies against LC3 (#4108; 1 : 1,000; Cell Signaling Technology, Inc., Danvers, MA,USA), p-mTOR (#2971, Cell Signaling Technology, Inc., Danvers, MA, USA), and mTOR (#2972, Cell Signaling Technology, Inc., Danvers, MA, USA) at 4°C overnight. .. Afterward, the membranes were incubated with a horseradish peroxidase-conjugated secondary antibody at room temperature for 1 h. An enhanced chemiluminescent autoradiography was adopted to detect immunoreactivity based on manufacturer's instructions.

Incubation:

Article Title: Neural Differentiation of Human Adipose Tissue-Derived Stem Cells Involves Activation of the Wnt5a/JNK Signalling
Article Snippet: .. After blocking with 1% skim milk (Sigma Chemical Co.) in PBS-T, the membranes were incubated with specific antibodies for Wnt3a (Cell Signaling Technology, Inc., Danvers, MA, USA, 1 : 1,000), Wnt5a/b (Cell Signaling Technology, 1 : 4,000), Dvl2 (Cell Signaling Technology, 1 : 1,000), Naked1 (Cell Signaling Technology, 1 : 1,000), Axin 1 (Cell Signaling Technology, 1 : 2,000), phosphor-JNK (Santa Cruz Biotechnology, Santa Cruz, CA, USA, 1 : 4,000), JNK (Santa Cruz Biotechnology, 1 : 4,000), phosphor-ERK1/2 (Santa Cruz Biotechnology, 1 : 1,000), ERK1/2 (Santa Cruz Biotechnology, 1 : 4,000), phosphor-PKC (Santa Cruz Biotechnology, 1 : 4,000), PKC (Santa Cruz Biotechnology, 1 : 1,000), phosphor-GSK3β (Cell Signaling Technology, 1 : 3,000), GSK3β (Cell Signaling Technology, 1 : 3,000), β -catenin (Cell Signaling Technology, 1 : 4,000), or GAPDH (Santa Cruz Biotechnology, 1 : 4,000). .. Following several washes, membranes were subsequently incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (Cell Signaling Technology).

Article Title: Activation of Akt/protein kinase B mediates the protective effects of mechanical stretching against myocardial ischemia-reperfusion injury
Article Snippet: .. Next, the membranes were incubated overnight with mouse monoclonal anti-phospho-Akt and anti-phospho-GSK-3β antibodies (1 : 1,000; Cell Signaling Technology, USA), or anti-Akt and anti-GSK-3β antibodies (1 : 1,000; Cell Signaling Technology, USA) in 5% skim milk and Tris-buffered saline at 4℃. .. After washing three times with Tris-buffered saline (15 min per wash), the blots were incubated with a goat anti-rat IgG antiserum conjugated to horseradish peroxidase (1 : 1,000; Santa Cruz Biotechnology, USA) for 2 h at 25℃.

Article Title: Long-Zhi Decoction Medicated Serum Promotes Angiogenesis in Human Umbilical Vein Endothelial Cells Based on Autophagy
Article Snippet: .. The membrane was blocked with a commercial protein blocking solution for 1 h after the blotting procedure, followed by washing three times with 1x Tris-buffered saline (TBS, 20 mM Tris-HCl, 136 mM NaCl, pH 7.6) containing 1% Tween-20 for 10 min. Then, the membrane was incubated overnight at 4°C with anti-LC3 (1 : 1,000, Cell Signaling Technology, USA), anti-Beclin-1 (1 : 1,000, Abcam, UK), and Anti-β -Actin (1 : 2,000, Santa Cruz Biotechnology, Inc., USA). .. On the next day, the membrane was washed three times with TBS for 15 min, followed by incubation with Goat Anti-Rabbit IgG (H + L) antibody (1 : 15,000, Cell Signaling Technology, USA) for 1 h at room temperature.

Article Title: Effects of Astragalus Polysaccharides on Dysfunction of Mitochondrial Dynamics Induced by Oxidative Stress
Article Snippet: .. The membranes were incubated with primary antibodies including anti-PGC-1, anti-MnSOD, anti-p53, and anti-GAPDH (1 : 1,000; Santa Cruz Biotechnology) as well as anti-Atg7, anti-p62, and anti-LC3 (1 : 1,000; Cell Signaling) at 4°C overnight. .. Then, the membranes were incubated with anti-rabbit or anti-mouse antibodies at room temperature for 1 h. The protein bands were captured and documented through a CCD system (Image Station 2000MM, Kodak, Rochester, NY, USA) or a gel image analysis system (ChemiDox XRS, Bio-Rad, USA).

Article Title: Study on the Mechanism of mTOR-Mediated Autophagy during Electroacupuncture Pretreatment against Cerebral Ischemic Injury
Article Snippet: .. A 60 μ g aliquot of proteins from each sample was extracted by applying 10% SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane, which was then incubated with the specific antibodies against LC3 (#4108; 1 : 1,000; Cell Signaling Technology, Inc., Danvers, MA,USA), p-mTOR (#2971, Cell Signaling Technology, Inc., Danvers, MA, USA), and mTOR (#2972, Cell Signaling Technology, Inc., Danvers, MA, USA) at 4°C overnight. .. Afterward, the membranes were incubated with a horseradish peroxidase-conjugated secondary antibody at room temperature for 1 h. An enhanced chemiluminescent autoradiography was adopted to detect immunoreactivity based on manufacturer's instructions.

Whole Genome Amplification:

Article Title: Fatty Acid Oxidation Promotes Cardiomyocyte Proliferation Rate but Does Not Change Cardiomyocyte Number in Infant Mice
Article Snippet: .. Primary antibodies are: Ki67 (1:50; Abcam, ab16667), Phospho-Histone H3 (PH3, 1:200; Cell Signaling Technology; 9706L), Aurora B kinase (1:200; BD Transduction Laboratories; 611082), cardiac Troponin T (cTnT, 1:100; Thermo Fisher Scientific, MS-295-P1), cardiac Troponin I (cTnI, 1:200; Abcam; ab47003), Caveolin-1 (1:100, Cell Signaling Technology; 3238S), Wheat Germ Agglutinin (WGA), Alexa Fluor® 633 Conjugate was used on the same sections to outline cardiomyocytes. ..

Blocking Assay:

Article Title: Neural Differentiation of Human Adipose Tissue-Derived Stem Cells Involves Activation of the Wnt5a/JNK Signalling
Article Snippet: .. After blocking with 1% skim milk (Sigma Chemical Co.) in PBS-T, the membranes were incubated with specific antibodies for Wnt3a (Cell Signaling Technology, Inc., Danvers, MA, USA, 1 : 1,000), Wnt5a/b (Cell Signaling Technology, 1 : 4,000), Dvl2 (Cell Signaling Technology, 1 : 1,000), Naked1 (Cell Signaling Technology, 1 : 1,000), Axin 1 (Cell Signaling Technology, 1 : 2,000), phosphor-JNK (Santa Cruz Biotechnology, Santa Cruz, CA, USA, 1 : 4,000), JNK (Santa Cruz Biotechnology, 1 : 4,000), phosphor-ERK1/2 (Santa Cruz Biotechnology, 1 : 1,000), ERK1/2 (Santa Cruz Biotechnology, 1 : 4,000), phosphor-PKC (Santa Cruz Biotechnology, 1 : 4,000), PKC (Santa Cruz Biotechnology, 1 : 1,000), phosphor-GSK3β (Cell Signaling Technology, 1 : 3,000), GSK3β (Cell Signaling Technology, 1 : 3,000), β -catenin (Cell Signaling Technology, 1 : 4,000), or GAPDH (Santa Cruz Biotechnology, 1 : 4,000). .. Following several washes, membranes were subsequently incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (Cell Signaling Technology).

Article Title: Long-Zhi Decoction Medicated Serum Promotes Angiogenesis in Human Umbilical Vein Endothelial Cells Based on Autophagy
Article Snippet: .. The membrane was blocked with a commercial protein blocking solution for 1 h after the blotting procedure, followed by washing three times with 1x Tris-buffered saline (TBS, 20 mM Tris-HCl, 136 mM NaCl, pH 7.6) containing 1% Tween-20 for 10 min. Then, the membrane was incubated overnight at 4°C with anti-LC3 (1 : 1,000, Cell Signaling Technology, USA), anti-Beclin-1 (1 : 1,000, Abcam, UK), and Anti-β -Actin (1 : 2,000, Santa Cruz Biotechnology, Inc., USA). .. On the next day, the membrane was washed three times with TBS for 15 min, followed by incubation with Goat Anti-Rabbit IgG (H + L) antibody (1 : 15,000, Cell Signaling Technology, USA) for 1 h at room temperature.

Mass Spectrometry:

Article Title: Fatty Acid Oxidation Promotes Cardiomyocyte Proliferation Rate but Does Not Change Cardiomyocyte Number in Infant Mice
Article Snippet: .. Primary antibodies are: Ki67 (1:50; Abcam, ab16667), Phospho-Histone H3 (PH3, 1:200; Cell Signaling Technology; 9706L), Aurora B kinase (1:200; BD Transduction Laboratories; 611082), cardiac Troponin T (cTnT, 1:100; Thermo Fisher Scientific, MS-295-P1), cardiac Troponin I (cTnI, 1:200; Abcam; ab47003), Caveolin-1 (1:100, Cell Signaling Technology; 3238S), Wheat Germ Agglutinin (WGA), Alexa Fluor® 633 Conjugate was used on the same sections to outline cardiomyocytes. ..

Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 92
    Cell Signaling Technology Inc nfatc1
    TRAIL inhibits RANKL-induced osteoclast differentiation and activation of <t>NFATc1.</t> a Bone marrow-derived macrophages (BMMs) from wild type (WT) and TRAIL-R knockout ( Trail-r −/− ) mice were plated in 96-well plates and stimulated with the RANKL (50 ng/ml) + M-CSF (20 ng/ml), TRAIL (500 ng/ml), or RANKL + M-CSF + TRAIL as indicated in the figure. After 10 days, cells were analyzed for osteoclast differentiation. After incubation, cells were subjected to a tartrate-resistant acid phosphatase (TRAP) assay. Cell morphology was examined by light microscopy (Scale bars, 100 µm), and the number of TRAP-positive multinuclear cells was quantified in ( b ). ** p
    Nfatc1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 92/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/nfatc1/product/Cell Signaling Technology Inc
    Average 92 stars, based on 7 article reviews
    Price from $9.99 to $1999.99
    nfatc1 - by Bioz Stars, 2020-09
    92/100 stars
      Buy from Supplier

    Image Search Results


    TRAIL inhibits RANKL-induced osteoclast differentiation and activation of NFATc1. a Bone marrow-derived macrophages (BMMs) from wild type (WT) and TRAIL-R knockout ( Trail-r −/− ) mice were plated in 96-well plates and stimulated with the RANKL (50 ng/ml) + M-CSF (20 ng/ml), TRAIL (500 ng/ml), or RANKL + M-CSF + TRAIL as indicated in the figure. After 10 days, cells were analyzed for osteoclast differentiation. After incubation, cells were subjected to a tartrate-resistant acid phosphatase (TRAP) assay. Cell morphology was examined by light microscopy (Scale bars, 100 µm), and the number of TRAP-positive multinuclear cells was quantified in ( b ). ** p

    Journal: Cell Death & Disease

    Article Title: TRAIL inhibits RANK signaling and suppresses osteoclast activation via inhibiting lipid raft assembly and TRAF6 recruitment

    doi: 10.1038/s41419-019-1353-3

    Figure Lengend Snippet: TRAIL inhibits RANKL-induced osteoclast differentiation and activation of NFATc1. a Bone marrow-derived macrophages (BMMs) from wild type (WT) and TRAIL-R knockout ( Trail-r −/− ) mice were plated in 96-well plates and stimulated with the RANKL (50 ng/ml) + M-CSF (20 ng/ml), TRAIL (500 ng/ml), or RANKL + M-CSF + TRAIL as indicated in the figure. After 10 days, cells were analyzed for osteoclast differentiation. After incubation, cells were subjected to a tartrate-resistant acid phosphatase (TRAP) assay. Cell morphology was examined by light microscopy (Scale bars, 100 µm), and the number of TRAP-positive multinuclear cells was quantified in ( b ). ** p

    Article Snippet: To further confirm whether RANKL or TRAIL induces osteoclastogenesis signaling through nuclear translocation of NFATc1, the critical transcription factor of osteoclasts, we isolated nuclei to detect the translocation of NFATc1 when cells were treated with RANKL plus M-CSF in the presence or absence of TRAIL.

    Techniques: Activation Assay, Derivative Assay, Knock-Out, Mouse Assay, Incubation, TRAP Assay, Light Microscopy

    Hypericin inhibits breast cancer-induced osteoclast differentiation and function via suppression of the NFATc1 signaling pathway and attenuation of Ca 2+ oscillation in osteoclasts ( A ) RAW264.7 cells (3 × 10 3 cells/well) were incubated in the presence of MCF-7 or MDA-MB-231 cells for 24 h, exposed to HP (1 μmol/L) for 5 days, and finally stained for measurement of TRAP expression. ( B ) Multinucleated osteoclasts ( > 3 nuclei) in co-cultures were counted. Columns represent the mean results of experiments carried out in triplicate, whereas bars represent the SD. ( C ) HP-inhibited osteoclast bone resorption induced by tumor cells. RAW264.7 cells (3 × 10 3 cells/well) were seeded into bovine bone slices in the presence of MCF-7 or MDA-MB-231 cells for 24 h, and treated with HP (1 μmol/L) for 5 days. After 5 days of incubation, images were obtained using a scanning electron microscope (SEM). Images of bone resorption pits are shown. ( D ) Resorption pit areas were measured using ImageJ software. Columns represent the mean results of experiments carried out in triplicate, whereas bars represent the SD. ( E ) RAW264.7 cells were incubated in serum-free media containing the indicated concentrations of HP and RANKL for 24 h. The cells were lysed, and total RNA was subjected to RT-PCR for determination of NFATc1 gene expression. Graphs indicate the relative intensity of NFATc1 compared to that of GAPDH. ( F ) RAW264.7 cells that were stably transfected with a NFATc1 luciferase reporter construct were pretreated with the indicated concentrations of HP for 1 h and then incubated in the absence or presence of RANKL for 12 h. Luciferase activity was then determined using the Promega luciferase assay system. ( G ) RANKL-induced NFATc1 translocation to the nucleus was assessed by western blotting. RAW264.7 cells (1 × 10 6 cells/well) were pretreated with the indicated concentrations of HP for 2 h and then stimulated with RANKL (50 ng/mL) or were untreated (controls) for 15 min. Cell nuclear extracts were prepared and subjected to western blotting using anti-NFATc1 and LaminA/C. Cell cytosol extracts were prepared and subjected to western blotting using anti-phospho-NFATc1 and actin. ( H ) Effects of HP on nuclear translocation of NFATc1 in RAW264.7 cells. Cells were treated with RANKL for 72 h in the presence and absence of HP (1 mmol/L) and stained with anti-NFATc1 antibody to investigate NFATc1 nuclear translocation (left panel). Nuclei were stained with DAPI (middle panel). Merged images of NFATc1 and the nuclei are shown in the right panel. ( I ) HP reduces intracellular Ca 2+ levels and calcium influx. RAW264.7 cells (3 × 10 3 cells/well) were incubated with RANKL (100 ng/mL) in the presence or absence of HP (1 μM) for 72 h. For Ca 2+ measurement, cells were incubated with Fluo-4 AM and 0.05% pluronic F-127 (Invitrogen) in HBSS supplemented with 1% FCS and 1 mM probenecid (assay buffer) for 30 min followed by confocal analysis. Representative fluo-4 fluorescent images of the RAW264.7 cells from different treatment groups are shown. Pseudo-color-labeled (purple) area represents the cells that are actively undergoing fluorescence ratio changes. ( J ) The relative intracellular Ca 2+ levels in individual cells were monitored for 5 min at 5-second intervals using the fluorescence intensity of Fluo-4 at 200× magnification. Cells with at least two oscillations were counted as oscillating cells. A minimum of 40 cells were monitored in triplicate wells. The average amplitude of Ca 2+ oscillations in each cell was calculated using the TuneR and SeeWave packages for the R programming language. Representative traces of three randomly chosen BMMs were recorded in different treatment groups. The fluorescence ratio change was recorded every 5 s for 300 s.

    Journal: Oncotarget

    Article Title: Hypericin targets osteoclast and prevents breast cancer-induced bone metastasis via NFATc1 signaling pathway

    doi: 10.18632/oncotarget.22930

    Figure Lengend Snippet: Hypericin inhibits breast cancer-induced osteoclast differentiation and function via suppression of the NFATc1 signaling pathway and attenuation of Ca 2+ oscillation in osteoclasts ( A ) RAW264.7 cells (3 × 10 3 cells/well) were incubated in the presence of MCF-7 or MDA-MB-231 cells for 24 h, exposed to HP (1 μmol/L) for 5 days, and finally stained for measurement of TRAP expression. ( B ) Multinucleated osteoclasts ( > 3 nuclei) in co-cultures were counted. Columns represent the mean results of experiments carried out in triplicate, whereas bars represent the SD. ( C ) HP-inhibited osteoclast bone resorption induced by tumor cells. RAW264.7 cells (3 × 10 3 cells/well) were seeded into bovine bone slices in the presence of MCF-7 or MDA-MB-231 cells for 24 h, and treated with HP (1 μmol/L) for 5 days. After 5 days of incubation, images were obtained using a scanning electron microscope (SEM). Images of bone resorption pits are shown. ( D ) Resorption pit areas were measured using ImageJ software. Columns represent the mean results of experiments carried out in triplicate, whereas bars represent the SD. ( E ) RAW264.7 cells were incubated in serum-free media containing the indicated concentrations of HP and RANKL for 24 h. The cells were lysed, and total RNA was subjected to RT-PCR for determination of NFATc1 gene expression. Graphs indicate the relative intensity of NFATc1 compared to that of GAPDH. ( F ) RAW264.7 cells that were stably transfected with a NFATc1 luciferase reporter construct were pretreated with the indicated concentrations of HP for 1 h and then incubated in the absence or presence of RANKL for 12 h. Luciferase activity was then determined using the Promega luciferase assay system. ( G ) RANKL-induced NFATc1 translocation to the nucleus was assessed by western blotting. RAW264.7 cells (1 × 10 6 cells/well) were pretreated with the indicated concentrations of HP for 2 h and then stimulated with RANKL (50 ng/mL) or were untreated (controls) for 15 min. Cell nuclear extracts were prepared and subjected to western blotting using anti-NFATc1 and LaminA/C. Cell cytosol extracts were prepared and subjected to western blotting using anti-phospho-NFATc1 and actin. ( H ) Effects of HP on nuclear translocation of NFATc1 in RAW264.7 cells. Cells were treated with RANKL for 72 h in the presence and absence of HP (1 mmol/L) and stained with anti-NFATc1 antibody to investigate NFATc1 nuclear translocation (left panel). Nuclei were stained with DAPI (middle panel). Merged images of NFATc1 and the nuclei are shown in the right panel. ( I ) HP reduces intracellular Ca 2+ levels and calcium influx. RAW264.7 cells (3 × 10 3 cells/well) were incubated with RANKL (100 ng/mL) in the presence or absence of HP (1 μM) for 72 h. For Ca 2+ measurement, cells were incubated with Fluo-4 AM and 0.05% pluronic F-127 (Invitrogen) in HBSS supplemented with 1% FCS and 1 mM probenecid (assay buffer) for 30 min followed by confocal analysis. Representative fluo-4 fluorescent images of the RAW264.7 cells from different treatment groups are shown. Pseudo-color-labeled (purple) area represents the cells that are actively undergoing fluorescence ratio changes. ( J ) The relative intracellular Ca 2+ levels in individual cells were monitored for 5 min at 5-second intervals using the fluorescence intensity of Fluo-4 at 200× magnification. Cells with at least two oscillations were counted as oscillating cells. A minimum of 40 cells were monitored in triplicate wells. The average amplitude of Ca 2+ oscillations in each cell was calculated using the TuneR and SeeWave packages for the R programming language. Representative traces of three randomly chosen BMMs were recorded in different treatment groups. The fluorescence ratio change was recorded every 5 s for 300 s.

    Article Snippet: Primary antibodies against β-actin, phospho-nuclear factor of activated T-cells 1 (phospho-NFATc1), and NFATc1 were purchased from Cell Signaling Technology (USA).

    Techniques: Incubation, Multiple Displacement Amplification, Staining, Expressing, Microscopy, Software, Reverse Transcription Polymerase Chain Reaction, Stable Transfection, Transfection, Luciferase, Construct, Activity Assay, Translocation Assay, Western Blot, Labeling, Fluorescence

    Adenovirus-mediated CXCR2 siRNA inhibited RANKL-induced osteoclast-specific gene and protein expression in vitro . ( A ) The mRNA expression of RANK , TRAP , CtsK , and c-Fos was significantly decreased in the CXCR2 group compared with the MS and control groups. ( B ) Western blotting revealed that TRAP, CtsK, and NFATc1 levels were decreased in the CXCR2 group. There were no other significant differences between the MS and control groups. All experiments were performed at least 3 times. * P

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    Article Title: Adenovirus-Mediated siRNA Targeting CXCR2 Attenuates Titanium Particle-Induced Osteolysis by Suppressing Osteoclast Formation

    doi: 10.12659/MSM.897243

    Figure Lengend Snippet: Adenovirus-mediated CXCR2 siRNA inhibited RANKL-induced osteoclast-specific gene and protein expression in vitro . ( A ) The mRNA expression of RANK , TRAP , CtsK , and c-Fos was significantly decreased in the CXCR2 group compared with the MS and control groups. ( B ) Western blotting revealed that TRAP, CtsK, and NFATc1 levels were decreased in the CXCR2 group. There were no other significant differences between the MS and control groups. All experiments were performed at least 3 times. * P

    Article Snippet: Anti-TRAP and -CXCR2 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and RANKL, TRAP, CtsK, NFATc1, and OPG antibodies were purchased from Cell Signaling Technology (Beverly, MA).

    Techniques: Expressing, In Vitro, Mass Spectrometry, Western Blot