nfatc1 signalling Search Results


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  • 99
    Millipore polyvinylidene difluoride membranes
    Polyvinylidene Difluoride Membranes, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 34311 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Santa Cruz Biotechnology nfatc1
    Expression profiles of transcription factors and osteoporosis PCR arrays in untransfected and <t>NFATc1-knockdown</t> RAW 264.7 cells. PCR arrays for transcription factors and osteoporosis were performed. The cluster heat maps for the expression of all genes from ( A ) transcription factors and ( B ) osteoporosis PCR arrays were shown for untransfected cells (left) and NFATc1-knockdown cells (right). The red squares represent up-regulated genes, the green ones down-regulated genes, the black ones unchanged genes and the grey ones are technically unacceptable data. These latter were not considered in the analysis of our results. Differentially expressed genes between untransfected and NFATc1-knockdown cells, RANKL-induced. ( C ) Up- and down-regulated genes, ( D ) up-regulated genes, ( E ) down-regulated genes.
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    93
    GeneTex gapdh
    Diaph1 deletion improved calcium dynamics after I/R. (a) <t>SERCA2a/GAPDH</t> protein levels were increased by Diaph1 deletion in both mouse hearts after LAD/reperfusion (n = 10/group; *p
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    94
    Cell Signaling Technology Inc p38
    Dose‐dependent reduction of age‐related cardiac inflammation by phenolic compounds. (a) Representative microphotographs of left ventricular sections of the heart stained with hematoxylin‐eosin showing young, SHAM, DMSO, PC 2.5, PC 5, PC 10, and PC 20 groups. (b) Histograms showing cardiomyocytes width (μm) and semiquantitative scores of inflammation ( n = 6–8 per group). (c) CRP and IL‐6 plasma concentrations in all groups ( n = 6–8 per group). (d) Representative western blot analyses for <t>p38</t> in rat cardiac tissue, normalized to GAPDH (in arbitrary units, a.u.) ( n = 3 for each protein and condition). * p
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    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
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    95
    Cell Signaling Technology Inc erk1 2
    Sustained association of NFAT and ERK with the insulin gene promoter requires CN and <t>ERK1/2</t> activity. ChIP-qPCR time-course analysis of fold enrichment of NFATc2 and ERK1/2 upon the insulin gene promoter in (A) MIN6 cells and (B) human islets in response
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    91
    Santa Cruz Biotechnology anti nfatc1
    VEGF signaling mediated by VEGFR2 is not required for cell proliferation, cell survival, expression of FoxP1, or nuclear translocation of <t>NFATc1</t> in endocardial cells of elongating mitral valves (MVs). (A, B) Immunofluorescent staining for phospho-histone
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    92
    GeneTex β actin
    Expression level and number of motoneurons in wild-type and homozygous shaky mice. (A) Western blot analysis of GlyR α1 subunit protein expression in whole brain and spinal cord homogenates. Normalization with <t>β-actin</t> showed no significant differences of GlyR α1 protein levels between the two genotypes ( n = 3), right image shows an example of the Western blot from two different animals of each genotype ( Glra1 +/+ and Glra1 sh / sh ). (B) Quantitative analysis of motoneuron numbers. The quantification was made using 3–4 animals of each genotype ( Glra1 +/+ , n = 4; Glra1 sh / sh , n = 3). Right images are examples of brain stem sections from Glra1 +/+ and Glra1 sh / sh with arrow heads pointing to motoneurons. Error bars represent standard deviations (S.D.).
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    99
    Cell Signaling Technology Inc c fos
    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 <t>c-Fos</t> and <t>NFATc1</t> compared with treatment with control medium. **P
    C Fos, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1342 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc β actin
    Portulaca oleracea ethanol extract (POEE) attenuates receptor activator of NF-κB ligand (RANKL)-mediated cytotoxicity. a Bone marrow-derived macrophages (BMMs) in 96-well plates were stimulated with RANKL for the indicated time. Following incubation, glucose-6-phosphate dehydrogenase (G6PD) in the culture medium was measured. The amount of released G6PD is calculated as a percentage of total G6PD, and results are presented as relative mean fold change. b BMMs were cultured under the indicated conditions. Polyadenosine 5′-diphosphate-ribose polymerase (PARP) expression and cleavage were evaluated by performing western blot analysis. Summarized data are presented as a ratio of cleaved PARP/uncleaved PARP in lower panel. <t>β-actin</t> was used for loading control
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    90
    Cell Signaling Technology Inc nfatc2
    <t>STAT3-NFATc2</t> signaling pathway was involved in the SIRT3-mediated fibroblast transdifferention. A-D. Immunoblot analysis of NFATc2 in WT, SIRT3-KO and LV.SIRT3 myofibroblasts. GAPDH expression was used as loading control. The bar graph represents protein quantification measured by densitometry analysis. (n=5). E. Representative immunofluorescence images indicated the subcellular location of NFATc2 in WT and SIRT3-KO myofibroblasts. Nuclei was stained with DAPI. Scale bar: 20 μm. The data are presented as the means ± SEM of three independent experiments. *P
    Nfatc2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 90/100, based on 131 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore β actin
    Adiponectin-deficient calvarial cells preferentially differentiate into adipocytes. (A–C) Mouse calvarial cells were incubated with β-glycerophosphate (10 mM) and ascorbic acid (50 μM) to induce osteoblast differentiation. (A) The cells were stained for ALP at day 7 and with alizarin red S at day 21 (upper, whole-well image; lower, 40× magnification). (B) Alizarin red S-stained cells were dissolved in 20% methanol and 10% acetic acid and the optical density (OD) was measured at 450 nm. (C) At days 5 and 10, total RNAs were isolated, reverse-transcribed, and subjected to RT-PCR to determine the mRNA levels of BSP, OCN, ALP, β-catenin, Runx2, and <t>β-actin.</t> (D–F) Mouse calvarial cells were incubated with dexamethasone (1 μM), insulin (5 μg/ml), and IBMX (0.5 mM) for the evaluation of adipogenic potential. (D) At day 21, the cells were fixed and stained using oil red O (upper, whole-well image; lower, 40× magnification). (E) The oil red O-stained cells were dissolved in isopropanol and the OD was measured at 500 nm. (F) At day 5, total RNAs were isolated, reverse-transcribed, and subjected to RT-PCR. The ratio of PPARγ to β-actin was obtained by using a densitometer. Data are mean values ± SD of triplicate samples and are representative of three similar independent experiments. * P
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    93
    GeneTex rad51
    Survivin silencing impaired DNA repair by homologous recombination. a qPCR analysis of a set of genes involved in DNA damage repair in Cal51, MDAMB-231, and MCF7 cells depleted or not in Survivin. Data are presented as means (±sem) of ratios normalized to controls from three independent experiments. b HR activity was evaluated by GFP gene conversion assay using the genetically modified RG37 cell line. RG37 cells were first transfected with I-Sce-I coding plasmid, and 24 h later depleted in Survivin or BRCA1 using specific siRNA. After 48 h, % of GFP positive cells was assessed in each condition by flow cytometry and presented as means (±sem) from six independent experiments. c Breast cancer cell lines were depleted in Survivin (SiSurvivin) or not (SiCt) for 48 h and immunoblot analysis were performed to evaluate expression of MUS81/EME1 complex and <t>RAD51</t> proteins
    Rad51, supplied by GeneTex, used in various techniques. Bioz Stars score: 93/100, based on 227 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Cell Signaling Technology Inc gapdh
    NGA inhibited osteoclast gene expression in mouse BMMs in vitro . The expression of mRNAs corresponding to gene products specific to RANKL-induced osteoclast differentiation, including TRAP , CTR , CTSK , and <t>NFATc1</t> in mouse BMMs treated with 30 ng/ml M-CSF, 50 ng/ml RANKL, and increasing concentrations of NGA (0, 0.2 or 0.4 μg/ml) for 5 days, measured by real-time PCR. RNA expression levels were normalized relative to the expression of <t>GAPDH</t> . Gene expression after being treated with NGA, compared with the 0 μg/ml treatment group (t-test), * p
    Gapdh, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 30834 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Santa Cruz Biotechnology β actin
    Madecassoside ( MA ) inhibits the RANKL ‐induced MAPK signaling pathway. (A) Representative western blot images of p‐ JNK 1/2, JNK , p‐ ERK 1/2, p‐p38, p38, ERK , and <t>β‐actin</t> at 0, 10, 20, 30, 60 min stimulated by GST ‐ rRANKL (50 ng/mL) with or without MA (10 μmol L −1 ). (B‐F) The relative ratios of phosphorylated proteins to unphosphorylated proteins were quantitatively determined. The data in the figures represent the means ± SD . Significant differences between the treatment and control groups are indicated as * P
    β Actin, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 97/100, based on 50832 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    GeneTex α tubulin
    Effect of BBR on HDAC activity and acetylation of histone H3 and <t>α-tubulin.</t> (a) Total cells lysates were incubated with various doses of BBR or 5 μM SAHA in HDAC assay buffer for 3 h. HDAC activity was initiated by adding the HDAC substrate and incubating at 37°C for 1 h. HDAC activity was measured by detecting the OD value at 405 nm. (b) MDA-MB-231 cells were treated with 25 or 50 μM BBR for 24 ~ 72 h or 1 μM SAHA for 24 h. Protein expressions of Ac-histone H3, Ac-α-tubulin and β-actin were analyzed by Western blotting. Images of each indicated probe were cropped from the same blot. (c) MDA-MB-231 cells were treated with 50 μM BBR for 48 h, and nuclear and cytosolic extracts were prepared as described in “Methods”. Protein expressions of Ac-histone H3, histone H3, Ac-α-tubulin, α-tubulin, HDAC1, HDAC2, HDAC6, and GAPDH were analyzed by Western blotting. Images of each indicated probe were cropped from the same blot.
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    92
    Cell Signaling Technology Inc phospho erk
    rCD200 inhibits RANKL signaling pathway. (A and B) PBMC adherent cells were cultured with M-CSF with or without rCD200 for 48 hr and stimulated for different times with RANKL. Expression of total protein levels and of the phosphorylated forms of <t>p38,</t> <t>ERK,</t> c-fos and NFATc1 was determined by Western blotting. The results of 1 experiment, representative of 3, are presented. (C) rCD200 inhibits osteoclast-related gene expression. Cells were cultivated with M-CSF for 48 hr, RANKL was added on day 3 of culture and medium was changed every 3 days. rCD200 was added at the beginning of the culture and every 3 days. Expression of target gene was determined by real-time PCR after 21 days of culture. Data are mean±SD from 3 independent experiments. *, P
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    92
    Santa Cruz Biotechnology nfatc2
    Physical interaction at the NFAT target sequence. DNA oligonucleotide, which includes the NFAT consensus binding sequence GGAAA, was either incubated with the cell lysate of cells stimulated with Ionomycin or untreated cells ( a ) or transiently transfected with ‘Silencer Negative Control’—siRNA or <t>NFATc2-specific</t> siRNA oligonucleotide and also stimulated or not stimulated ( b ). Proteins binding to the biotin-marked oligonucleotide sequence were subsequently precipitated with streptavidin. By means of the respective antibodies, NFATc2 and Sp1 were determined by Western blot analysis
    Nfatc2, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 433 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Expression profiles of transcription factors and osteoporosis PCR arrays in untransfected and NFATc1-knockdown RAW 264.7 cells. PCR arrays for transcription factors and osteoporosis were performed. The cluster heat maps for the expression of all genes from ( A ) transcription factors and ( B ) osteoporosis PCR arrays were shown for untransfected cells (left) and NFATc1-knockdown cells (right). The red squares represent up-regulated genes, the green ones down-regulated genes, the black ones unchanged genes and the grey ones are technically unacceptable data. These latter were not considered in the analysis of our results. Differentially expressed genes between untransfected and NFATc1-knockdown cells, RANKL-induced. ( C ) Up- and down-regulated genes, ( D ) up-regulated genes, ( E ) down-regulated genes.

    Journal: Cells

    Article Title: Gene Expression Profiling of NFATc1-Knockdown in RAW 264.7 Cells: An Alternative Pathway for Macrophage Differentiation

    doi: 10.3390/cells8020131

    Figure Lengend Snippet: Expression profiles of transcription factors and osteoporosis PCR arrays in untransfected and NFATc1-knockdown RAW 264.7 cells. PCR arrays for transcription factors and osteoporosis were performed. The cluster heat maps for the expression of all genes from ( A ) transcription factors and ( B ) osteoporosis PCR arrays were shown for untransfected cells (left) and NFATc1-knockdown cells (right). The red squares represent up-regulated genes, the green ones down-regulated genes, the black ones unchanged genes and the grey ones are technically unacceptable data. These latter were not considered in the analysis of our results. Differentially expressed genes between untransfected and NFATc1-knockdown cells, RANKL-induced. ( C ) Up- and down-regulated genes, ( D ) up-regulated genes, ( E ) down-regulated genes.

    Article Snippet: These results suggest that MITF functions downstream of NFATc1 within RANKL signaling, even if it was found that MITF up-regulation was sufficient to induce osteoclast markers such as TRAP, DC-STAMP, CtsK and ATP6V0 [ ].

    Techniques: Expressing, Polymerase Chain Reaction

    Gene Ontology analysis as molecular function of untransfected and NFATc1-knockdown cells. Gene Ontology analysis as molecular function represented by pie chart ( A ) untransfected cells, RANKL-induced; and ( B ) NFATc1-knockdown cells, RANKL-induced.

    Journal: Cells

    Article Title: Gene Expression Profiling of NFATc1-Knockdown in RAW 264.7 Cells: An Alternative Pathway for Macrophage Differentiation

    doi: 10.3390/cells8020131

    Figure Lengend Snippet: Gene Ontology analysis as molecular function of untransfected and NFATc1-knockdown cells. Gene Ontology analysis as molecular function represented by pie chart ( A ) untransfected cells, RANKL-induced; and ( B ) NFATc1-knockdown cells, RANKL-induced.

    Article Snippet: These results suggest that MITF functions downstream of NFATc1 within RANKL signaling, even if it was found that MITF up-regulation was sufficient to induce osteoclast markers such as TRAP, DC-STAMP, CtsK and ATP6V0 [ ].

    Techniques:

    GATA2 is a new target of NFATc1. Cells were transfected with siRNA-NC and siRNA-NFATc1 for 24 and 72 h before protein expression levels were analyzed. ( A ) Western blot of NFATc1 and GATA2 after 24 and 72 h. ( B ) Western blot of NFATc1 and STAT6 after 24 h. β-actin was used as loading control. The data shown represent two independent experiments with comparable outcomes. ( C ) QPCR of Arginase 1 . ( D ) QPCR of IL-10, IL-13 and TNF-α . mRNA expression is presented as relative values to those expressed in siRNA-NC transfected cells arbitrarily set at 1.0. The results shown are the means ± SD of three experiments (each of which was performed in triplicate). * p

    Journal: Cells

    Article Title: Gene Expression Profiling of NFATc1-Knockdown in RAW 264.7 Cells: An Alternative Pathway for Macrophage Differentiation

    doi: 10.3390/cells8020131

    Figure Lengend Snippet: GATA2 is a new target of NFATc1. Cells were transfected with siRNA-NC and siRNA-NFATc1 for 24 and 72 h before protein expression levels were analyzed. ( A ) Western blot of NFATc1 and GATA2 after 24 and 72 h. ( B ) Western blot of NFATc1 and STAT6 after 24 h. β-actin was used as loading control. The data shown represent two independent experiments with comparable outcomes. ( C ) QPCR of Arginase 1 . ( D ) QPCR of IL-10, IL-13 and TNF-α . mRNA expression is presented as relative values to those expressed in siRNA-NC transfected cells arbitrarily set at 1.0. The results shown are the means ± SD of three experiments (each of which was performed in triplicate). * p

    Article Snippet: These results suggest that MITF functions downstream of NFATc1 within RANKL signaling, even if it was found that MITF up-regulation was sufficient to induce osteoclast markers such as TRAP, DC-STAMP, CtsK and ATP6V0 [ ].

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

    Effects of NFATc1 induction or depletion on different genes involved in the RANK–RANKL pathway. Cells were untransfected and RANKL-induced. QPCR results of ( A ) NFATc1, Myc and Acp5 ; ( B ) Jun, Esr1 and Egr 1; and ( C ) CtsK, Hnf1a, Smad5 and Stat4 . Cells were NFATc1-knockdown and RANKL-induced. QPCR results of ( D ) GATA2 and Runx2 ; ( E ) NFATc1, CtsK, Acp5, Stat4, Hnf1a and Egr1 ; ( F ) Smad5, Esr1 and Jun ; and ( G ) RhoA, DC-STAMP, MMP9, MITF, FOS and OSCAR . The mRNA expression levels were presented as relative values to those expressed in siRNA-NC transfected or untransfected cells arbitrarily set at 1.0. The results shown are the means ± SD of three experiments (each of which was performed in triplicate). * p

    Journal: Cells

    Article Title: Gene Expression Profiling of NFATc1-Knockdown in RAW 264.7 Cells: An Alternative Pathway for Macrophage Differentiation

    doi: 10.3390/cells8020131

    Figure Lengend Snippet: Effects of NFATc1 induction or depletion on different genes involved in the RANK–RANKL pathway. Cells were untransfected and RANKL-induced. QPCR results of ( A ) NFATc1, Myc and Acp5 ; ( B ) Jun, Esr1 and Egr 1; and ( C ) CtsK, Hnf1a, Smad5 and Stat4 . Cells were NFATc1-knockdown and RANKL-induced. QPCR results of ( D ) GATA2 and Runx2 ; ( E ) NFATc1, CtsK, Acp5, Stat4, Hnf1a and Egr1 ; ( F ) Smad5, Esr1 and Jun ; and ( G ) RhoA, DC-STAMP, MMP9, MITF, FOS and OSCAR . The mRNA expression levels were presented as relative values to those expressed in siRNA-NC transfected or untransfected cells arbitrarily set at 1.0. The results shown are the means ± SD of three experiments (each of which was performed in triplicate). * p

    Article Snippet: These results suggest that MITF functions downstream of NFATc1 within RANKL signaling, even if it was found that MITF up-regulation was sufficient to induce osteoclast markers such as TRAP, DC-STAMP, CtsK and ATP6V0 [ ].

    Techniques: Real-time Polymerase Chain Reaction, Expressing, Transfection

    Protein network analysis using Ingenuity Pathway Analysis (IPA). Proteins identified in the dataset are highlighted in red or in green when they exhibit a higher or a lower content in NFATc1-knockdown cells compared to untransfected cells. The intensity of the node color indicates the expression level or degree of regulation. Genes in uncolored notes were not differentially expressed in our experiment and were integrated into the computationally generated networks based on the evidence stored in the IPA knowledge memory, which indicated relevance to this network.

    Journal: Cells

    Article Title: Gene Expression Profiling of NFATc1-Knockdown in RAW 264.7 Cells: An Alternative Pathway for Macrophage Differentiation

    doi: 10.3390/cells8020131

    Figure Lengend Snippet: Protein network analysis using Ingenuity Pathway Analysis (IPA). Proteins identified in the dataset are highlighted in red or in green when they exhibit a higher or a lower content in NFATc1-knockdown cells compared to untransfected cells. The intensity of the node color indicates the expression level or degree of regulation. Genes in uncolored notes were not differentially expressed in our experiment and were integrated into the computationally generated networks based on the evidence stored in the IPA knowledge memory, which indicated relevance to this network.

    Article Snippet: These results suggest that MITF functions downstream of NFATc1 within RANKL signaling, even if it was found that MITF up-regulation was sufficient to induce osteoclast markers such as TRAP, DC-STAMP, CtsK and ATP6V0 [ ].

    Techniques: Indirect Immunoperoxidase Assay, Expressing, Generated

    Inhibition of osteoclastogenesis by silencing of NFATc1. Untransfected, siRNA-non correlated (NC) and siRNA-NFATc1 transfected cells were cultured with RANKL (50 ng/mL) for 24 h. Control untransfected cells were cultured without RANKL. ( A ) Cells were fixed, stained with DAPI (which stains the nuclei blue) and observed by DIC (upper row) and immunofluorescence (middle row) microscopy. Bottom row shows merged images. ( B ) Quantitative PCR (QPCR) of NFATc1 . mRNA expression was presented as relative values to those expressed in untransfected cells (−/+RANKL) arbitrarily set at 1.0. The results shown are the means ± SD of two experiments (each of which was performed in triplicate). *** p

    Journal: Cells

    Article Title: Gene Expression Profiling of NFATc1-Knockdown in RAW 264.7 Cells: An Alternative Pathway for Macrophage Differentiation

    doi: 10.3390/cells8020131

    Figure Lengend Snippet: Inhibition of osteoclastogenesis by silencing of NFATc1. Untransfected, siRNA-non correlated (NC) and siRNA-NFATc1 transfected cells were cultured with RANKL (50 ng/mL) for 24 h. Control untransfected cells were cultured without RANKL. ( A ) Cells were fixed, stained with DAPI (which stains the nuclei blue) and observed by DIC (upper row) and immunofluorescence (middle row) microscopy. Bottom row shows merged images. ( B ) Quantitative PCR (QPCR) of NFATc1 . mRNA expression was presented as relative values to those expressed in untransfected cells (−/+RANKL) arbitrarily set at 1.0. The results shown are the means ± SD of two experiments (each of which was performed in triplicate). *** p

    Article Snippet: These results suggest that MITF functions downstream of NFATc1 within RANKL signaling, even if it was found that MITF up-regulation was sufficient to induce osteoclast markers such as TRAP, DC-STAMP, CtsK and ATP6V0 [ ].

    Techniques: Inhibition, Transfection, Cell Culture, Staining, Immunofluorescence, Microscopy, Real-time Polymerase Chain Reaction, Expressing

    Gene Ontology analysis as biological function of untransfected and NFATc1-knockdown RAW 264.7 cells. ( A ) Untransfected cells, RANKL-induced. ( B ) NFATc1-knockdown cells, RANKL-induced. The count represents the number of genes showing variation in their expression levels involved in the annotated biological process. The p value denotes the significance of Gene Ontology (GO) term enrichment in the differentially expressed mRNAs based on the PANTHER classification system. This analysis allowed us to determine the biological pathways for which a significant enrichment of differentially expressed genes existed ( p

    Journal: Cells

    Article Title: Gene Expression Profiling of NFATc1-Knockdown in RAW 264.7 Cells: An Alternative Pathway for Macrophage Differentiation

    doi: 10.3390/cells8020131

    Figure Lengend Snippet: Gene Ontology analysis as biological function of untransfected and NFATc1-knockdown RAW 264.7 cells. ( A ) Untransfected cells, RANKL-induced. ( B ) NFATc1-knockdown cells, RANKL-induced. The count represents the number of genes showing variation in their expression levels involved in the annotated biological process. The p value denotes the significance of Gene Ontology (GO) term enrichment in the differentially expressed mRNAs based on the PANTHER classification system. This analysis allowed us to determine the biological pathways for which a significant enrichment of differentially expressed genes existed ( p

    Article Snippet: These results suggest that MITF functions downstream of NFATc1 within RANKL signaling, even if it was found that MITF up-regulation was sufficient to induce osteoclast markers such as TRAP, DC-STAMP, CtsK and ATP6V0 [ ].

    Techniques: Expressing

    Diaph1 deletion improved calcium dynamics after I/R. (a) SERCA2a/GAPDH protein levels were increased by Diaph1 deletion in both mouse hearts after LAD/reperfusion (n = 10/group; *p

    Journal: EBioMedicine

    Article Title: The Formin, DIAPH1, is a Key Modulator of Myocardial Ischemia/Reperfusion Injury

    doi: 10.1016/j.ebiom.2017.11.012

    Figure Lengend Snippet: Diaph1 deletion improved calcium dynamics after I/R. (a) SERCA2a/GAPDH protein levels were increased by Diaph1 deletion in both mouse hearts after LAD/reperfusion (n = 10/group; *p

    Article Snippet: We used primary antibodies (1:1000) as follows: mDia1 (DIAPH1), β-actin, ROCK2, nucleoporin p62 (BD Biosciences, San Jose, CA), pan-actin (Cytoskeleton, Denver, CO), GAPDH (Genetex, Irvine, CA), EGR1, SERCA2a (Santa Cruz, Dallas, TX), DIAPH1, myocardin, NCX1, calsequestrin, DIAPH2, histone 3, GAPDH (AbCam, Cambridge, UK), phosphorylated (ser16) and total phospholamban (EMD Millipore, Billerica, MA), GAPDH (Genetex, Irvine, CA), phosphorylated and total Rac1, RhoA, phosphorylated (ser9) and total GSK3β (Cell Signaling, Danvers, MA).

    Techniques:

    Reduction of COX-2 expression and catalytic activity by shRNA or NS398 reduce DENV replication. COX-2 shRNA reduced DENV replication in ( A ) DENV replicon cells and ( B to D ) a DENV infection system. Huh-7-D2-FLuc-SGR-Neo DENV replicon cells were transfected with GFP or COX-2 shRNA at the indicated concentrations for 3 days and the cell lysates were subjected to a luciferase activity assay and western blotting. Huh-7 cells were transfected with COX-2 shRNA at the indicated concentrations, and the transfected cells were infected with DENV-2 at an MOI of 1. After 3 days of treatment, the cell lysate, cellular RNA and supernatants were analyzed by western blotting, RT-qPCR or plaque assay, respectively. NS398 reduced DENV replication in ( E ) DENV replicon cells and ( F to H ) a DENV infection system. Huh-7-D2-FLuc-SGR-Neo DENV replicon cells were treated with NS398 at different concentrations (0, 5, 10, 20, and 40 μM) for 3 days, and the cell lysates were subjected to a luciferase activity assay and western blotting. Huh-7 cells were infected with DENV-2 at an MOI of 1 and then treated with NS398 at different concentrations (0, 5, 10, 20, and 40 μM) for 3 days. Western blotting was performed with anti-COX-2, anti-NS2B, and anti-GAPDH antibodies. The relative RNA level of DENV-2 was determined by RT-qPCR following normalization to the cellular gapdh mRNA level. All data are indicative of at least three independent experiments, with each measurement performed in triplicate. Error bars are expressed as the mean ± SD of three independent experiments; * P

    Journal: Scientific Reports

    Article Title: Cyclooxygenase‐2 facilitates dengue virus replication and serves as a potential target for developing antiviral agents

    doi: 10.1038/srep44701

    Figure Lengend Snippet: Reduction of COX-2 expression and catalytic activity by shRNA or NS398 reduce DENV replication. COX-2 shRNA reduced DENV replication in ( A ) DENV replicon cells and ( B to D ) a DENV infection system. Huh-7-D2-FLuc-SGR-Neo DENV replicon cells were transfected with GFP or COX-2 shRNA at the indicated concentrations for 3 days and the cell lysates were subjected to a luciferase activity assay and western blotting. Huh-7 cells were transfected with COX-2 shRNA at the indicated concentrations, and the transfected cells were infected with DENV-2 at an MOI of 1. After 3 days of treatment, the cell lysate, cellular RNA and supernatants were analyzed by western blotting, RT-qPCR or plaque assay, respectively. NS398 reduced DENV replication in ( E ) DENV replicon cells and ( F to H ) a DENV infection system. Huh-7-D2-FLuc-SGR-Neo DENV replicon cells were treated with NS398 at different concentrations (0, 5, 10, 20, and 40 μM) for 3 days, and the cell lysates were subjected to a luciferase activity assay and western blotting. Huh-7 cells were infected with DENV-2 at an MOI of 1 and then treated with NS398 at different concentrations (0, 5, 10, 20, and 40 μM) for 3 days. Western blotting was performed with anti-COX-2, anti-NS2B, and anti-GAPDH antibodies. The relative RNA level of DENV-2 was determined by RT-qPCR following normalization to the cellular gapdh mRNA level. All data are indicative of at least three independent experiments, with each measurement performed in triplicate. Error bars are expressed as the mean ± SD of three independent experiments; * P

    Article Snippet: The antibodies used in the present study included anti-DENV NS2B (1:3000; GeneTex, Irvine, CA, USA), anti-GAPDH (1:3000; GeneTex, Irvine, CA, USA), anti-COX-2 (1:1000, Cayman, ML, USA), and anti-Myc (1:2000; Abcam, Cambridge, MA, USA).

    Techniques: Expressing, Activity Assay, shRNA, Infection, Transfection, Luciferase, Western Blot, Quantitative RT-PCR, Plaque Assay

    COX-2 overexpression and PGE 2 treatment increase DENV-2 replication. COX-2 overexpression induced DENV-2 replication in ( A ) DENV-2 replicon cells and ( B and C ) the DENV infection system. Huh-7-D2-FLuc-SGR-Neo DENV replicon reporter cells were transfected with pcDNA4/Myc or pcDNA4-COX-2-Myc at the indicated concentrations. After 3 days of incubation, the cell lysates were subjected to a luciferase activity assay. Huh-7 cells were transfected with pcDNA4/Myc or pcDNA4-COX-2-Myc at the indicated concentrations, and the transfected cells were infected with DENV-2 at an MOI of 1. After 3 days of incubation, the cell lysates and cellular RNA were subjected to western blotting and RT-qPCR. ( D ) COX-2 overexpression increased DENV-2 propagation. The transfected Huh-7 cells were infected by DENV-2 at a MOI of 1 for 3 days. Supernatants were collected and subjected to a viral plaque assay. PGE 2 treatment induced DENV-2 replication in ( E ) viral replicon cells and ( F and G ) the DENV infection system. Huh-7-D2-FLuc-SGR-Neo DENV replicon reporter cells were treated with PGE 2 at the indicated concentrations for 3 days and the cell lysates were subjected to a luciferase activity assay. Huh-7 cells were infected with DENV-2 at an MOI of 1, and the infected cells were treated with PGE 2 at the indicated concentrations for 3 days. Western blotting was performed with anti-NS2B, anti-Myc, and anti-GAPDH antibodies. Relative RNA levels of DENV-2 was determined by RT-qPCR following the normalization of cellular gapdh mRNA levels. ( H ) PGE 2 treatment induced DENV-2 propagation. Huh-7 cells were infected with DENV-2 at an MOI of 1 and then treated with PGE 2 . Supernatants were collected and subjected to a viral plaque assay. ( I ) PGE 2 treatment induced DENV-2 NS5 polymerase activity. Huh-7 cells were cotransfected with p(+)RLuc-(−)DV-UTRΔC-Fluc reporter template (0.5 μg) and pcDNA-NS5-Myc expression plasmid (0.5 μg), and the transfected cells were treated with PGE 2 at the indicated concentrations for 3 days. The cell lysates were subjected to a Dual-Glo Luciferase Assay. All data were indicative of at least three independent experiments, with each measurement carried out in triplicate. Error bars are expressed as the mean ± SD of three independent experiments; * P

    Journal: Scientific Reports

    Article Title: Cyclooxygenase‐2 facilitates dengue virus replication and serves as a potential target for developing antiviral agents

    doi: 10.1038/srep44701

    Figure Lengend Snippet: COX-2 overexpression and PGE 2 treatment increase DENV-2 replication. COX-2 overexpression induced DENV-2 replication in ( A ) DENV-2 replicon cells and ( B and C ) the DENV infection system. Huh-7-D2-FLuc-SGR-Neo DENV replicon reporter cells were transfected with pcDNA4/Myc or pcDNA4-COX-2-Myc at the indicated concentrations. After 3 days of incubation, the cell lysates were subjected to a luciferase activity assay. Huh-7 cells were transfected with pcDNA4/Myc or pcDNA4-COX-2-Myc at the indicated concentrations, and the transfected cells were infected with DENV-2 at an MOI of 1. After 3 days of incubation, the cell lysates and cellular RNA were subjected to western blotting and RT-qPCR. ( D ) COX-2 overexpression increased DENV-2 propagation. The transfected Huh-7 cells were infected by DENV-2 at a MOI of 1 for 3 days. Supernatants were collected and subjected to a viral plaque assay. PGE 2 treatment induced DENV-2 replication in ( E ) viral replicon cells and ( F and G ) the DENV infection system. Huh-7-D2-FLuc-SGR-Neo DENV replicon reporter cells were treated with PGE 2 at the indicated concentrations for 3 days and the cell lysates were subjected to a luciferase activity assay. Huh-7 cells were infected with DENV-2 at an MOI of 1, and the infected cells were treated with PGE 2 at the indicated concentrations for 3 days. Western blotting was performed with anti-NS2B, anti-Myc, and anti-GAPDH antibodies. Relative RNA levels of DENV-2 was determined by RT-qPCR following the normalization of cellular gapdh mRNA levels. ( H ) PGE 2 treatment induced DENV-2 propagation. Huh-7 cells were infected with DENV-2 at an MOI of 1 and then treated with PGE 2 . Supernatants were collected and subjected to a viral plaque assay. ( I ) PGE 2 treatment induced DENV-2 NS5 polymerase activity. Huh-7 cells were cotransfected with p(+)RLuc-(−)DV-UTRΔC-Fluc reporter template (0.5 μg) and pcDNA-NS5-Myc expression plasmid (0.5 μg), and the transfected cells were treated with PGE 2 at the indicated concentrations for 3 days. The cell lysates were subjected to a Dual-Glo Luciferase Assay. All data were indicative of at least three independent experiments, with each measurement carried out in triplicate. Error bars are expressed as the mean ± SD of three independent experiments; * P

    Article Snippet: The antibodies used in the present study included anti-DENV NS2B (1:3000; GeneTex, Irvine, CA, USA), anti-GAPDH (1:3000; GeneTex, Irvine, CA, USA), anti-COX-2 (1:1000, Cayman, ML, USA), and anti-Myc (1:2000; Abcam, Cambridge, MA, USA).

    Techniques: Over Expression, Infection, Transfection, Incubation, Luciferase, Activity Assay, Western Blot, Quantitative RT-PCR, Viral Plaque Assay, Expressing, Plasmid Preparation

    COX-2 expression is required for viral replication. ( A to C ) Exogenous COX-2 expression restored DENV-2 protein synthesis, RNA replication and viral propagation in COX-2 shRNA-transfected cells. Huh-7 cells were cotransfected with COX-2 shRNA (1.0 μg) and pCMV-COX-2-Myc (0.25, 0.5, and 1.0 μg), followed by DENV-2 infection at an MOI of 1. ( D to F ) Exogenous COX-2 expression restored DENV-2 protein synthesis, RNA replication and viral propagation in NS398-treated cells. Huh-7 cells were transfected with pcDNA4/Myc (0.5 μg) or pCMV-COX-2-Myc (0.25, 0.5, and 1.0 μg), followed by DENV-2 infection at an MOI of 1. The cells were treated with DMSO or 40 μM NS398 for 3 days. Western blotting was performed with anti-NS2B, anti-Myc, and anti-GAPDH antibodies. The relative RNA level of DENV-2 was determined by RT-qPCR following normalization to the cellular gapdh mRNA level. All data are indicative of at least three independent experiments, with each measurement performed in triplicate. Error bars are expressed as the mean ± SD of three independent experiments; * P

    Journal: Scientific Reports

    Article Title: Cyclooxygenase‐2 facilitates dengue virus replication and serves as a potential target for developing antiviral agents

    doi: 10.1038/srep44701

    Figure Lengend Snippet: COX-2 expression is required for viral replication. ( A to C ) Exogenous COX-2 expression restored DENV-2 protein synthesis, RNA replication and viral propagation in COX-2 shRNA-transfected cells. Huh-7 cells were cotransfected with COX-2 shRNA (1.0 μg) and pCMV-COX-2-Myc (0.25, 0.5, and 1.0 μg), followed by DENV-2 infection at an MOI of 1. ( D to F ) Exogenous COX-2 expression restored DENV-2 protein synthesis, RNA replication and viral propagation in NS398-treated cells. Huh-7 cells were transfected with pcDNA4/Myc (0.5 μg) or pCMV-COX-2-Myc (0.25, 0.5, and 1.0 μg), followed by DENV-2 infection at an MOI of 1. The cells were treated with DMSO or 40 μM NS398 for 3 days. Western blotting was performed with anti-NS2B, anti-Myc, and anti-GAPDH antibodies. The relative RNA level of DENV-2 was determined by RT-qPCR following normalization to the cellular gapdh mRNA level. All data are indicative of at least three independent experiments, with each measurement performed in triplicate. Error bars are expressed as the mean ± SD of three independent experiments; * P

    Article Snippet: The antibodies used in the present study included anti-DENV NS2B (1:3000; GeneTex, Irvine, CA, USA), anti-GAPDH (1:3000; GeneTex, Irvine, CA, USA), anti-COX-2 (1:1000, Cayman, ML, USA), and anti-Myc (1:2000; Abcam, Cambridge, MA, USA).

    Techniques: Expressing, shRNA, Transfection, Infection, Western Blot, Quantitative RT-PCR

    DENV induces COX-2 expression and PGE 2 production in DF patients, DENV-infected mice, and human hepatoma cells. ( A and B ) Elevated COX-2 expression and PGE 2 levels in the blood of dengue fever patients. COX-2 mRNA and PGE 2 levels in blood samples from 13 clinical DF patients and 6 healthy donors were determined by RT-qPCR or ELISA, respectively. ( C ) The induced COX-2 expression in DENV-2-infected ICR suckling mice. Six-day-old suckling mice were injected with 2.5 × 10 5 pfu of DENV-2 or heat-inactivated DENV-2 (iDENV) by intracerebral injection. Each group comprised six suckling mice (n = 6). Six days after inoculation, COX-2 mRNA levels of mouse brain tissues were determined by RT-qPCR. DENV-2 time-dependently induced ( D ) COX-2 protein expression, ( E ) COX-2 RNA replication, and ( F ) PGE 2 production. Huh-7 cells were infected with DENV-2 at an MOI of 0.1, and the cell lysate and cellular RNA were extracted at the indicated time points (24, 48, and 72 hpi). Western blotting was performed with anti-COX-2, anti-NS2B, and anti-GAPDH antibodies. Relative RNA levels of DENV-2 and COX-2 were determined by RT-qPCR following the normalization of cellular gapdh mRNA levels. Supernatants were collected at the indicated time points and subjected to a PGE 2 ELISA assay. All data from cell-based experiments are indicative of at least three independent experiments, with each measurement carried out in triplicate. Error bars are expressed as the mean ± SD of three independent experiments; * P

    Journal: Scientific Reports

    Article Title: Cyclooxygenase‐2 facilitates dengue virus replication and serves as a potential target for developing antiviral agents

    doi: 10.1038/srep44701

    Figure Lengend Snippet: DENV induces COX-2 expression and PGE 2 production in DF patients, DENV-infected mice, and human hepatoma cells. ( A and B ) Elevated COX-2 expression and PGE 2 levels in the blood of dengue fever patients. COX-2 mRNA and PGE 2 levels in blood samples from 13 clinical DF patients and 6 healthy donors were determined by RT-qPCR or ELISA, respectively. ( C ) The induced COX-2 expression in DENV-2-infected ICR suckling mice. Six-day-old suckling mice were injected with 2.5 × 10 5 pfu of DENV-2 or heat-inactivated DENV-2 (iDENV) by intracerebral injection. Each group comprised six suckling mice (n = 6). Six days after inoculation, COX-2 mRNA levels of mouse brain tissues were determined by RT-qPCR. DENV-2 time-dependently induced ( D ) COX-2 protein expression, ( E ) COX-2 RNA replication, and ( F ) PGE 2 production. Huh-7 cells were infected with DENV-2 at an MOI of 0.1, and the cell lysate and cellular RNA were extracted at the indicated time points (24, 48, and 72 hpi). Western blotting was performed with anti-COX-2, anti-NS2B, and anti-GAPDH antibodies. Relative RNA levels of DENV-2 and COX-2 were determined by RT-qPCR following the normalization of cellular gapdh mRNA levels. Supernatants were collected at the indicated time points and subjected to a PGE 2 ELISA assay. All data from cell-based experiments are indicative of at least three independent experiments, with each measurement carried out in triplicate. Error bars are expressed as the mean ± SD of three independent experiments; * P

    Article Snippet: The antibodies used in the present study included anti-DENV NS2B (1:3000; GeneTex, Irvine, CA, USA), anti-GAPDH (1:3000; GeneTex, Irvine, CA, USA), anti-COX-2 (1:1000, Cayman, ML, USA), and anti-Myc (1:2000; Abcam, Cambridge, MA, USA).

    Techniques: Expressing, Infection, Mouse Assay, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Injection, Western Blot

    NF-κB and MAPK/JNK-mediated C/EBP are responsible for DENV-2-induced COX-2 expression and viral replication. ( A ) DENV-2 induced activation of the NF-κB signaling pathway. Huh-7 cells were infected by DENV-2 at an MOI of 1 and the cell lysates were extracted at the indicated time points. Western blotting was performed and the relative blot intensities were quantified by densitometry scanning. ( B ) CAPE significantly suppressed DENV-2-induced COX-2 expression. Huh-7 cells were pretreated with DMSO or CAPE (20 μM) for 2 h and then infected with DENV-2 at an MOI of 1. The cell lysates were analyzed at 2 dpi by western blotting. ( C and D ) CAPE dose-dependently suppressed DENV-2 protein synthesis and RNA replication. Huh-7 cells were infected by DENV-2 and treated with CAPE at different concentrations (0, 5, 10, and 20 μM). After 3 days of treatment, the cell lysates and cellular RNA were analyzed by western blotting or RT-qPCR, respectively. ( E ) DENV-2 induced the activation of the MAPK/JNK pathway. Huh-7 cells were infected by DENV-2, and the cell lysates were extracted at the indicated time points. Western blotting was performed and the relative blot intensities were quantified by densitometry scanning. ( F ) SP600125 significantly suppressed DENV-2-induced COX-2 expression. Huh-7 cells were pretreated with DMSO or SP600125 (20 μM) for 2 h, and then, the cells were infected by DENV-2. After 2 days of treatment, cell lysates were subjected to western blotting. ( G and H ) SP600125 dose-dependently suppressed DENV-2 protein synthesis and RNA replication. Huh-7 cells were infected by DENV-2, and the cells were treated with DMSO or SP600125 at different concentrations (0, 5, 10, and 20 μM). After 3 days of treatment, the cell lysates and cellular RNA were analyzed by western blotting or RT-qPCR, respectively. GAPDH served as a loading control in western blotting. The relative RNA level of DENV-2 was determine by RT-qPCR following normalization to the cellular gapdh mRNA level. All data are indicative of at least three independent experiments, with each measurement carried out in triplicate. Error bars are expressed as the mean ± SD of three independent experiments; * P

    Journal: Scientific Reports

    Article Title: Cyclooxygenase‐2 facilitates dengue virus replication and serves as a potential target for developing antiviral agents

    doi: 10.1038/srep44701

    Figure Lengend Snippet: NF-κB and MAPK/JNK-mediated C/EBP are responsible for DENV-2-induced COX-2 expression and viral replication. ( A ) DENV-2 induced activation of the NF-κB signaling pathway. Huh-7 cells were infected by DENV-2 at an MOI of 1 and the cell lysates were extracted at the indicated time points. Western blotting was performed and the relative blot intensities were quantified by densitometry scanning. ( B ) CAPE significantly suppressed DENV-2-induced COX-2 expression. Huh-7 cells were pretreated with DMSO or CAPE (20 μM) for 2 h and then infected with DENV-2 at an MOI of 1. The cell lysates were analyzed at 2 dpi by western blotting. ( C and D ) CAPE dose-dependently suppressed DENV-2 protein synthesis and RNA replication. Huh-7 cells were infected by DENV-2 and treated with CAPE at different concentrations (0, 5, 10, and 20 μM). After 3 days of treatment, the cell lysates and cellular RNA were analyzed by western blotting or RT-qPCR, respectively. ( E ) DENV-2 induced the activation of the MAPK/JNK pathway. Huh-7 cells were infected by DENV-2, and the cell lysates were extracted at the indicated time points. Western blotting was performed and the relative blot intensities were quantified by densitometry scanning. ( F ) SP600125 significantly suppressed DENV-2-induced COX-2 expression. Huh-7 cells were pretreated with DMSO or SP600125 (20 μM) for 2 h, and then, the cells were infected by DENV-2. After 2 days of treatment, cell lysates were subjected to western blotting. ( G and H ) SP600125 dose-dependently suppressed DENV-2 protein synthesis and RNA replication. Huh-7 cells were infected by DENV-2, and the cells were treated with DMSO or SP600125 at different concentrations (0, 5, 10, and 20 μM). After 3 days of treatment, the cell lysates and cellular RNA were analyzed by western blotting or RT-qPCR, respectively. GAPDH served as a loading control in western blotting. The relative RNA level of DENV-2 was determine by RT-qPCR following normalization to the cellular gapdh mRNA level. All data are indicative of at least three independent experiments, with each measurement carried out in triplicate. Error bars are expressed as the mean ± SD of three independent experiments; * P

    Article Snippet: The antibodies used in the present study included anti-DENV NS2B (1:3000; GeneTex, Irvine, CA, USA), anti-GAPDH (1:3000; GeneTex, Irvine, CA, USA), anti-COX-2 (1:1000, Cayman, ML, USA), and anti-Myc (1:2000; Abcam, Cambridge, MA, USA).

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

    Dose‐dependent reduction of age‐related cardiac inflammation by phenolic compounds. (a) Representative microphotographs of left ventricular sections of the heart stained with hematoxylin‐eosin showing young, SHAM, DMSO, PC 2.5, PC 5, PC 10, and PC 20 groups. (b) Histograms showing cardiomyocytes width (μm) and semiquantitative scores of inflammation ( n = 6–8 per group). (c) CRP and IL‐6 plasma concentrations in all groups ( n = 6–8 per group). (d) Representative western blot analyses for p38 in rat cardiac tissue, normalized to GAPDH (in arbitrary units, a.u.) ( n = 3 for each protein and condition). * p

    Journal: Aging Cell

    Article Title: Long‐term intake of phenolic compounds attenuates age‐related cardiac remodeling, et al. Long‐term intake of phenolic compounds attenuates age‐related cardiac remodeling

    doi: 10.1111/acel.12894

    Figure Lengend Snippet: Dose‐dependent reduction of age‐related cardiac inflammation by phenolic compounds. (a) Representative microphotographs of left ventricular sections of the heart stained with hematoxylin‐eosin showing young, SHAM, DMSO, PC 2.5, PC 5, PC 10, and PC 20 groups. (b) Histograms showing cardiomyocytes width (μm) and semiquantitative scores of inflammation ( n = 6–8 per group). (c) CRP and IL‐6 plasma concentrations in all groups ( n = 6–8 per group). (d) Representative western blot analyses for p38 in rat cardiac tissue, normalized to GAPDH (in arbitrary units, a.u.) ( n = 3 for each protein and condition). * p

    Article Snippet: The blots were then incubated overnight with gentle agitation at 4°C with primary antibodies: NFATc3 (Santa‐Cruz Biotechnology, Dallas, TX, USA; 1/500), phospho‐Ser165 NFATc3 (p‐NFATc3) (Abcam; 1/500), PP2B‐Aβ (C‐20) (Santa‐Cruz Biotechnology; 1/1,000), SOD1 (Abcam; 1/2000), SOD2 (Abcam; 1/4,000), Smad2 (Abcam; 1/500), phospho‐S467 Smad2 (p‐Smad2) (Abcam; 1/500), Smad3 (Abcam; 1/1,000), phospho‐S423+S425 Smad3 (p‐Smad3) (Abcam; 1/1,000), pan‐CAMKII (Cell Signaling Technology, MA, USA; 1/1,000), pan‐phospho CAMKII (Cell Signaling Technology; 1/1,000), troponin I (C‐4) (Santa‐Cruz Biotechnology; 1/500), GSK 3β (Cell Signaling Technology; 1/1,000), phospho‐GSK 3β (Cell Signaling Technology; 1/1,000), ERK1/2 (Abcam; 1/1,000), phospho‐ERK 1/2 (Abcam; 1/10,000), P38 (Cell Signaling Technology; 1/1,000), phospho‐Thr180/Tyr182 P38 (Cell Signaling Technology; 1/1,000), NF‐ƙB p65 (Ser536) (Cell Signaling Technology; 1/1,000), phospho‐NF‐ƙB p65 (Ser536) (93H1) (Cell Signaling Technology; 1/1,000), and glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) (Abcam; 1/2,500 for all).

    Techniques: Staining, Western Blot

    Western blot analysis of phosphorylated and total Akt protein in WT and TEAD-1 Tg hearts. Total protein extracts (100 μg) from the hearts of 10-month-old WT and TEAD-1 Tg (line 12) mice revealed no significant changes in total Akt and phospho-Akt

    Journal: The Journal of Biological Chemistry

    Article Title: TEAD-1 Overexpression in the Mouse Heart Promotes an Age-dependent Heart Dysfunction *

    doi: 10.1074/jbc.M109.063057

    Figure Lengend Snippet: Western blot analysis of phosphorylated and total Akt protein in WT and TEAD-1 Tg hearts. Total protein extracts (100 μg) from the hearts of 10-month-old WT and TEAD-1 Tg (line 12) mice revealed no significant changes in total Akt and phospho-Akt

    Article Snippet: The antibodies used in this study are as follows: TEAD-1 (BD Transduction Laboratories); HA (Cell Signaling); Akt, p-Akt, Akt1, and Akt2 (Cell Signaling); GSK-3α/β (Santa Cruz Biotechnology); p-Ser-GSK-3α and p-Ser-GSK-3β (Cell Signaling); glyceraldehyde-3-phosphate dehydrogenase and β-catenin (Cell Signaling); NFATc3 and NFATc4 (Santa Cruz Biotechnology); histone H1 (Santa Cruz Biotechnology); IP90 anti-peptide antibody (Abcam); and horseradish peroxidase-linked anti-rabbit IgG and anti-mouse IgG (Cell Signaling).

    Techniques: Western Blot, Mouse Assay

    Tetrandrine inhibited RANKL-induced NF-kB, MAPK, and PI3K/AKT pathways activation. Bone marrow monocytes (BMMs) were treated with M-CSF (20 ng/ml), RANKL (50 ng/ml), and tetrandrine (1 μM) and lysed at 0, 15, 30, and 60 min. The proteins were collected to detect the levels of phosphorylation of key proteins by Western blotting. (A) Phosphorylation of key proteins in NF-kB pathways, including P50, P65, and IκBα, were detected by Western blotting. (B) P65 immunofluorescence images were captured by fluorescence microscopy to detect the inhibitory effect of tetrandrine on RANKL-induced P65 nuclear translocation. (C) The nuclear/cytoplasmic fluorescence ratios were detected by image J. (D) Phosphorylation of key proteins in MAPK pathways, including ERK1/2, JNK, and, P38, were detected by Western blotting. (E) Phosphorylation of key proteins in PI3K-AKT pathways, including PI3K and AKT, were detected by Western blotting. **P

    Journal: Frontiers in Pharmacology

    Article Title: Tetrandrine Prevents Bone Loss in Ovariectomized Mice by Inhibiting RANKL-Induced Osteoclastogenesis

    doi: 10.3389/fphar.2019.01530

    Figure Lengend Snippet: Tetrandrine inhibited RANKL-induced NF-kB, MAPK, and PI3K/AKT pathways activation. Bone marrow monocytes (BMMs) were treated with M-CSF (20 ng/ml), RANKL (50 ng/ml), and tetrandrine (1 μM) and lysed at 0, 15, 30, and 60 min. The proteins were collected to detect the levels of phosphorylation of key proteins by Western blotting. (A) Phosphorylation of key proteins in NF-kB pathways, including P50, P65, and IκBα, were detected by Western blotting. (B) P65 immunofluorescence images were captured by fluorescence microscopy to detect the inhibitory effect of tetrandrine on RANKL-induced P65 nuclear translocation. (C) The nuclear/cytoplasmic fluorescence ratios were detected by image J. (D) Phosphorylation of key proteins in MAPK pathways, including ERK1/2, JNK, and, P38, were detected by Western blotting. (E) Phosphorylation of key proteins in PI3K-AKT pathways, including PI3K and AKT, were detected by Western blotting. **P

    Article Snippet: Primary antibodies against NFATc1, P-PI3K, AKT, P-AKT, P50, P-P50, P65, P-P65, IκBα, P-IκBα, ERK1/2, P-ERK1/2, JNK, P-JNK, P38, and P-P38 were obtained from Cell Signaling Technologies (Beverly, MA, USA).

    Techniques: Activation Assay, Western Blot, Immunofluorescence, Fluorescence, Microscopy, Translocation Assay

    Effect of VOS on MAPK/ATF2 signaling activation. a RAW264.7 cells were pretreated with VOS for 6 h and then co-treated with LPS (1 μg/ml) for 20 min. b RAW264.7 cells were pretreated with VOS for 6 h and then co-treated with LPS (1 μg/ml) for 30 min. After the treatment, the nucleus fraction was prepared. For Western blot analysis, the cell lysates were subjected to SDS-PAGE and the Western blot was performed using antibodies against p-ERK1/2, p-p38, p-JNK p-ATF2 and ATF2. Total-ERK1/2, total-p38 and total-JNK and actin were used as internal control for Western blot analysis. The density of Western blot bands was calculated using the software UN-SCAN-IT gel version 5.1 (Silk Scientific Inc. Orem, UT, USA). * P

    Journal: BMC Complementary and Alternative Medicine

    Article Title: Anti-inflammatory effect of Vaccinium oldhamii stems through inhibition of NF-κB and MAPK/ATF2 signaling activation in LPS-stimulated RAW264.7 cells

    doi: 10.1186/s12906-019-2720-4

    Figure Lengend Snippet: Effect of VOS on MAPK/ATF2 signaling activation. a RAW264.7 cells were pretreated with VOS for 6 h and then co-treated with LPS (1 μg/ml) for 20 min. b RAW264.7 cells were pretreated with VOS for 6 h and then co-treated with LPS (1 μg/ml) for 30 min. After the treatment, the nucleus fraction was prepared. For Western blot analysis, the cell lysates were subjected to SDS-PAGE and the Western blot was performed using antibodies against p-ERK1/2, p-p38, p-JNK p-ATF2 and ATF2. Total-ERK1/2, total-p38 and total-JNK and actin were used as internal control for Western blot analysis. The density of Western blot bands was calculated using the software UN-SCAN-IT gel version 5.1 (Silk Scientific Inc. Orem, UT, USA). * P

    Article Snippet: Antibodies against iNOS (#13120), COX-2 (#12282), IκB-α (#4814), p65 (#8242), phospho-ERK1/2 (#4377), ERK1/2 (#9102), phospho-p38 (#4511), p38 (#9212), phospho-JNK (#4668), JNK (#9258), p-ATF2 (#9221), ATF2 (#35031) and β-actin (#5125) were purchased from Cell Signaling (Bervely, MA, USA).

    Techniques: Activation Assay, Western Blot, SDS Page, Software

    RA specifically inhibited SRC/AKT signaling pathways during osteoclastogenesis. a RAW264.7 cells were treated with or without RANKL (50 ng/mL), RA (0.8 μM), or SC79 (25 μM) for 0, 10, or 30 min, respectively. Western blotting for p-AKT was analyzed with the cell lysates. b RAW264.7 cells were treated with or without RANKL (50 ng/mL), RA (0.8 μM), or SC79 (25 μM) for 0, 1, 3, or 5 days respectively. Western blotting for SRC was analyzed with the cell lysates. c RAW264.7 cells were treated with or without RA (0.8 μM) with the addition of RANKL (50 ng/mL) for 0, 10 or 30 min, respectively. Western blotting for MAPK and IκBα signaling pathways was analyzed with the cell lysates

    Journal: Cell Death & Disease

    Article Title: Raddeanin A suppresses breast cancer-associated osteolysis through inhibiting osteoclasts and breast cancer cells

    doi: 10.1038/s41419-018-0417-0

    Figure Lengend Snippet: RA specifically inhibited SRC/AKT signaling pathways during osteoclastogenesis. a RAW264.7 cells were treated with or without RANKL (50 ng/mL), RA (0.8 μM), or SC79 (25 μM) for 0, 10, or 30 min, respectively. Western blotting for p-AKT was analyzed with the cell lysates. b RAW264.7 cells were treated with or without RANKL (50 ng/mL), RA (0.8 μM), or SC79 (25 μM) for 0, 1, 3, or 5 days respectively. Western blotting for SRC was analyzed with the cell lysates. c RAW264.7 cells were treated with or without RA (0.8 μM) with the addition of RANKL (50 ng/mL) for 0, 10 or 30 min, respectively. Western blotting for MAPK and IκBα signaling pathways was analyzed with the cell lysates

    Article Snippet: Specific antibodies targeting SRC, ERK, JNK, p38, IκBα, phospho-IκBα, phospho-ERK, phospho-JNK, phospho-p38, phospho-AKT, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were obtained from Cell Signalling Technology (Cambridge, MA, USA).

    Techniques: Western Blot

    Ca- and Pi-induced activation of NF-kB, Erk1/2 and p38 MAP kinase, and STAT3 signaling is responsible for the MMP-3 and MMP-13 expression in hypertrophic chondrocytes. ( A ) Murine limb-bud MPCs were cultured at a high density (2.5 × 10 5 cells/10 μL) for 12 days and then treated with Ca (2.5 mM) or Pi (1.5 mM) for 5 mins. Total cell lysates were subjected to Western blotting for Erk1/2, p38, JNK MAP kinase, Akt, and NF-kB signaling. ( B , C ) Hypertrophic chondrocytes obtained from micromass cultures were treated with Ca and Pi for the indicated time course, and the phosphorylation of the components in each signaling pathway was assessed by Western blotting. n = 3. ( D ) Representative Western blot analysis of whole cell lysates from micromass cultures treated with specific inhibitors of Erk1/2 (PD98059), p38 MAP kinase (SB203058), NF-kB (JSH-23) and calcineurin-NFAT signaling (FK506) during the co-treatment with Ca and Pi for 24 h. ( E ) Micromass-cultured cells were treated with various doses of Stattic, a small-molecule inhibitor of STAT3 activation and dimerization. n = 3.

    Journal: Scientific Reports

    Article Title: Calcium-phosphate complex increased during subchondral bone remodeling affects earlystage osteoarthritis

    doi: 10.1038/s41598-017-18946-y

    Figure Lengend Snippet: Ca- and Pi-induced activation of NF-kB, Erk1/2 and p38 MAP kinase, and STAT3 signaling is responsible for the MMP-3 and MMP-13 expression in hypertrophic chondrocytes. ( A ) Murine limb-bud MPCs were cultured at a high density (2.5 × 10 5 cells/10 μL) for 12 days and then treated with Ca (2.5 mM) or Pi (1.5 mM) for 5 mins. Total cell lysates were subjected to Western blotting for Erk1/2, p38, JNK MAP kinase, Akt, and NF-kB signaling. ( B , C ) Hypertrophic chondrocytes obtained from micromass cultures were treated with Ca and Pi for the indicated time course, and the phosphorylation of the components in each signaling pathway was assessed by Western blotting. n = 3. ( D ) Representative Western blot analysis of whole cell lysates from micromass cultures treated with specific inhibitors of Erk1/2 (PD98059), p38 MAP kinase (SB203058), NF-kB (JSH-23) and calcineurin-NFAT signaling (FK506) during the co-treatment with Ca and Pi for 24 h. ( E ) Micromass-cultured cells were treated with various doses of Stattic, a small-molecule inhibitor of STAT3 activation and dimerization. n = 3.

    Article Snippet: The primary antibodies against Col2, Col10, Mmp-3, Mmp-13, Adamts5 and Runx2 were purchased from Abcam (Cambridge, UK); the phospho-Erk1/2, phospho-P38, phospho-JNK, phospho-Akt, phospho-P65, phospho-IkB, NFAT1 and NFAT3 antibodies were purchased from Cell Signaling Technology (Danvers, MA); and the Epas1 and NFAT4 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

    Techniques: Activation Assay, Expressing, Cell Culture, Western Blot

    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

    Sustained association of NFAT and ERK with the insulin gene promoter requires CN and ERK1/2 activity. ChIP-qPCR time-course analysis of fold enrichment of NFATc2 and ERK1/2 upon the insulin gene promoter in (A) MIN6 cells and (B) human islets in response

    Journal: Molecular Endocrinology

    Article Title: NFAT Targets Signaling Molecules to Gene Promoters in Pancreatic β-Cells

    doi: 10.1210/me.2014-1066

    Figure Lengend Snippet: Sustained association of NFAT and ERK with the insulin gene promoter requires CN and ERK1/2 activity. ChIP-qPCR time-course analysis of fold enrichment of NFATc2 and ERK1/2 upon the insulin gene promoter in (A) MIN6 cells and (B) human islets in response

    Article Snippet: These results indicate that NFAT activation, nuclear translocation, and DNA-binding are critical components of CN/NFAT signaling to target ERK1/2 to the insulin gene promoter.

    Techniques: Activity Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    CN/NFAT signaling is required for ERK1/2 to translocate to the insulin gene promoter in response to glucose and GLP-1. A, Effect of shRNA targeting of NFAT family isoforms on insulin gene promoter activity in response to high (G 16.7mM) glucose and 20nM

    Journal: Molecular Endocrinology

    Article Title: NFAT Targets Signaling Molecules to Gene Promoters in Pancreatic β-Cells

    doi: 10.1210/me.2014-1066

    Figure Lengend Snippet: CN/NFAT signaling is required for ERK1/2 to translocate to the insulin gene promoter in response to glucose and GLP-1. A, Effect of shRNA targeting of NFAT family isoforms on insulin gene promoter activity in response to high (G 16.7mM) glucose and 20nM

    Article Snippet: These results indicate that NFAT activation, nuclear translocation, and DNA-binding are critical components of CN/NFAT signaling to target ERK1/2 to the insulin gene promoter.

    Techniques: shRNA, Activity Assay

    Schematic model for NFAT-mediated activation and repression of insulin and TNF-α genes in β-cells in response to GLP-1 and IL-1β. GLP-1 in the presence of glucose activates CN/NFAT and ERK1/2 to induce enrichment of ERK-p300 and

    Journal: Molecular Endocrinology

    Article Title: NFAT Targets Signaling Molecules to Gene Promoters in Pancreatic β-Cells

    doi: 10.1210/me.2014-1066

    Figure Lengend Snippet: Schematic model for NFAT-mediated activation and repression of insulin and TNF-α genes in β-cells in response to GLP-1 and IL-1β. GLP-1 in the presence of glucose activates CN/NFAT and ERK1/2 to induce enrichment of ERK-p300 and

    Article Snippet: These results indicate that NFAT activation, nuclear translocation, and DNA-binding are critical components of CN/NFAT signaling to target ERK1/2 to the insulin gene promoter.

    Techniques: Activation Assay

    VEGF signaling mediated by VEGFR2 is not required for cell proliferation, cell survival, expression of FoxP1, or nuclear translocation of NFATc1 in endocardial cells of elongating mitral valves (MVs). (A, B) Immunofluorescent staining for phospho-histone

    Journal: Developmental biology

    Article Title: VEGF Signaling has Distinct Spatiotemporal Roles During Heart Valve Development

    doi: 10.1016/j.ydbio.2010.08.030

    Figure Lengend Snippet: VEGF signaling mediated by VEGFR2 is not required for cell proliferation, cell survival, expression of FoxP1, or nuclear translocation of NFATc1 in endocardial cells of elongating mitral valves (MVs). (A, B) Immunofluorescent staining for phospho-histone

    Article Snippet: For anti-Sox9 (Santa Cruz Biotechnology, Santa Cruz, CA) and anti-NFATc1 (Santa Cruz Biotechnology), sections were boiled for 10 minutes in 0.3M citrate, pH 6.0 buffer (Vector Laboratories, Burlingame, CA).

    Techniques: Expressing, Translocation Assay, Staining

    Expression level and number of motoneurons in wild-type and homozygous shaky mice. (A) Western blot analysis of GlyR α1 subunit protein expression in whole brain and spinal cord homogenates. Normalization with β-actin showed no significant differences of GlyR α1 protein levels between the two genotypes ( n = 3), right image shows an example of the Western blot from two different animals of each genotype ( Glra1 +/+ and Glra1 sh / sh ). (B) Quantitative analysis of motoneuron numbers. The quantification was made using 3–4 animals of each genotype ( Glra1 +/+ , n = 4; Glra1 sh / sh , n = 3). Right images are examples of brain stem sections from Glra1 +/+ and Glra1 sh / sh with arrow heads pointing to motoneurons. Error bars represent standard deviations (S.D.).

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Functional Consequences of the Postnatal Switch From Neonatal to Mutant Adult Glycine Receptor α1 Subunits in the Shaky Mouse Model of Startle Disease

    doi: 10.3389/fnmol.2018.00167

    Figure Lengend Snippet: Expression level and number of motoneurons in wild-type and homozygous shaky mice. (A) Western blot analysis of GlyR α1 subunit protein expression in whole brain and spinal cord homogenates. Normalization with β-actin showed no significant differences of GlyR α1 protein levels between the two genotypes ( n = 3), right image shows an example of the Western blot from two different animals of each genotype ( Glra1 +/+ and Glra1 sh / sh ). (B) Quantitative analysis of motoneuron numbers. The quantification was made using 3–4 animals of each genotype ( Glra1 +/+ , n = 4; Glra1 sh / sh , n = 3). Right images are examples of brain stem sections from Glra1 +/+ and Glra1 sh / sh with arrow heads pointing to motoneurons. Error bars represent standard deviations (S.D.).

    Article Snippet: GlyR proteins were detected with the GlyR α1 specific antibody mAb2b (cat. no. 146003 1:1,500, Synaptic Systems, Göttingen, Germany). β-Actin (cat. no. GTX26276, WB 1:5,000, GeneTex/Biozol, Eching, Germany) served as loading control.

    Techniques: Expressing, Mouse Assay, Western Blot

    Backcross of shaky mouse line into the spasmodic mouse line. Developmental expression of the GlyR α1 subunit in shaky mice and after backcross into the mouse line spasmodic (A–D) . After backcross of shaky into the spasmodic line, the expression profile was determined in spinal cord (sc), brain stem (bs) and cortex (cx) for stages P0, P7, P14, P28, P56, P84, and P100. (A) Glra1 +/+ , (B) Glra1 sh / spd , (C) heterozygous Glra1 +/ spd , and (D) heterozygous Glra1 +/ sh . Cortex (cx) served as negative control for GlyR α1. GlyR α1 subunit was stained with mAb2b (48 kDa), and β-Actin (46 kDa) served as a loading control (LC). Cortex samples were probed with a GlyR pan-α antibody (mAb4a) labeling other GlyR α subunits in the cortex (A,B) .

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Functional Consequences of the Postnatal Switch From Neonatal to Mutant Adult Glycine Receptor α1 Subunits in the Shaky Mouse Model of Startle Disease

    doi: 10.3389/fnmol.2018.00167

    Figure Lengend Snippet: Backcross of shaky mouse line into the spasmodic mouse line. Developmental expression of the GlyR α1 subunit in shaky mice and after backcross into the mouse line spasmodic (A–D) . After backcross of shaky into the spasmodic line, the expression profile was determined in spinal cord (sc), brain stem (bs) and cortex (cx) for stages P0, P7, P14, P28, P56, P84, and P100. (A) Glra1 +/+ , (B) Glra1 sh / spd , (C) heterozygous Glra1 +/ spd , and (D) heterozygous Glra1 +/ sh . Cortex (cx) served as negative control for GlyR α1. GlyR α1 subunit was stained with mAb2b (48 kDa), and β-Actin (46 kDa) served as a loading control (LC). Cortex samples were probed with a GlyR pan-α antibody (mAb4a) labeling other GlyR α subunits in the cortex (A,B) .

    Article Snippet: GlyR proteins were detected with the GlyR α1 specific antibody mAb2b (cat. no. 146003 1:1,500, Synaptic Systems, Göttingen, Germany). β-Actin (cat. no. GTX26276, WB 1:5,000, GeneTex/Biozol, Eching, Germany) served as loading control.

    Techniques: Expressing, Mouse Assay, Negative Control, Staining, Labeling

    Genotype of shaky mice. (A) The recognition site for the restriction enzyme HpyCH4V is disrupted by the shaky mouse mutation in exon 6; left panel. PCR genotyping of shaky ( Glra1 sh / sh ), wild-type ( Glra1 +/+ ) and heterozygous ( Glra1 +/ sh ) mice with subsequent HpyCH4V digest (right panel). (B) Sequencing chromatograms of wild-type strains C57BL6 and 129/SvJ, heterozygous Glra1 +/ sh , and homozygous Glra1 sh / sh showing a c.T198C transition in exon 3 and a c.C613A transition in exon 6. Both wild-type mouse strains are shown since the shaky mutation arose in a hybrid background of C57BL6 and 129/SvJ. (C) RT-PCR analysis of GlyR α1 subunit mRNA levels in spinal cord (sc) and brain stem (bs) of wild-type ( n = 4) and shaky mice ( n = 4). β-actin cDNA was amplified as a reference gene to ensure equal cDNA content in all samples.

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Functional Consequences of the Postnatal Switch From Neonatal to Mutant Adult Glycine Receptor α1 Subunits in the Shaky Mouse Model of Startle Disease

    doi: 10.3389/fnmol.2018.00167

    Figure Lengend Snippet: Genotype of shaky mice. (A) The recognition site for the restriction enzyme HpyCH4V is disrupted by the shaky mouse mutation in exon 6; left panel. PCR genotyping of shaky ( Glra1 sh / sh ), wild-type ( Glra1 +/+ ) and heterozygous ( Glra1 +/ sh ) mice with subsequent HpyCH4V digest (right panel). (B) Sequencing chromatograms of wild-type strains C57BL6 and 129/SvJ, heterozygous Glra1 +/ sh , and homozygous Glra1 sh / sh showing a c.T198C transition in exon 3 and a c.C613A transition in exon 6. Both wild-type mouse strains are shown since the shaky mutation arose in a hybrid background of C57BL6 and 129/SvJ. (C) RT-PCR analysis of GlyR α1 subunit mRNA levels in spinal cord (sc) and brain stem (bs) of wild-type ( n = 4) and shaky mice ( n = 4). β-actin cDNA was amplified as a reference gene to ensure equal cDNA content in all samples.

    Article Snippet: GlyR proteins were detected with the GlyR α1 specific antibody mAb2b (cat. no. 146003 1:1,500, Synaptic Systems, Göttingen, Germany). β-Actin (cat. no. GTX26276, WB 1:5,000, GeneTex/Biozol, Eching, Germany) served as loading control.

    Techniques: Mouse Assay, Mutagenesis, Polymerase Chain Reaction, Sequencing, Reverse Transcription Polymerase Chain Reaction, Amplification

    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

    Journal: Molecular Medicine Reports

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

    doi: 10.3892/mmr.2016.6041

    Figure Lengend 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

    Article Snippet: The membranes were blocked with PBS-Tween containing 5% non-fat skim milk for 2 h. The membranes were subsequently incubated with rabbit anti-human monoclonal antibodies against IκB, p-IκB, c-Fos, NFATc1, β-actin and GAPDH (dilution, 1:1,000), followed by incubation with the secondary horseradish peroxidase-conjugated anti-rabbit IgG antibody (cat. no. 7074; dilution, 1:5,000; Cell Signaling Technology, Inc.).

    Techniques: 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

    Journal: Molecular Medicine Reports

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

    doi: 10.3892/mmr.2016.6041

    Figure Lengend 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

    Article Snippet: The membranes were blocked with PBS-Tween containing 5% non-fat skim milk for 2 h. The membranes were subsequently incubated with rabbit anti-human monoclonal antibodies against IκB, p-IκB, c-Fos, NFATc1, β-actin and GAPDH (dilution, 1:1,000), followed by incubation with the secondary horseradish peroxidase-conjugated anti-rabbit IgG antibody (cat. no. 7074; dilution, 1:5,000; Cell Signaling Technology, Inc.).

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

    Portulaca oleracea ethanol extract (POEE) attenuates receptor activator of NF-κB ligand (RANKL)-mediated cytotoxicity. a Bone marrow-derived macrophages (BMMs) in 96-well plates were stimulated with RANKL for the indicated time. Following incubation, glucose-6-phosphate dehydrogenase (G6PD) in the culture medium was measured. The amount of released G6PD is calculated as a percentage of total G6PD, and results are presented as relative mean fold change. b BMMs were cultured under the indicated conditions. Polyadenosine 5′-diphosphate-ribose polymerase (PARP) expression and cleavage were evaluated by performing western blot analysis. Summarized data are presented as a ratio of cleaved PARP/uncleaved PARP in lower panel. β-actin was used for loading control

    Journal: BMC Complementary and Alternative Medicine

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

    doi: 10.1186/s12906-015-0770-9

    Figure Lengend Snippet: Portulaca oleracea ethanol extract (POEE) attenuates receptor activator of NF-κB ligand (RANKL)-mediated cytotoxicity. a Bone marrow-derived macrophages (BMMs) in 96-well plates were stimulated with RANKL for the indicated time. Following incubation, glucose-6-phosphate dehydrogenase (G6PD) in the culture medium was measured. The amount of released G6PD is calculated as a percentage of total G6PD, and results are presented as relative mean fold change. b BMMs were cultured under the indicated conditions. Polyadenosine 5′-diphosphate-ribose polymerase (PARP) expression and cleavage were evaluated by performing western blot analysis. Summarized data are presented as a ratio of cleaved PARP/uncleaved PARP in lower panel. β-actin was used for loading control

    Article Snippet: Antibodies against polyadenosine 5′-diphosphate-ribose polymerase (PARP), NFATc1, and β-actin were purchased from Cell Signaling Technology (MA, USA), Santa Cruz Biotechnology (TX, USA), and Sigma Aldrich (MO, USA), respectively.

    Techniques: Derivative Assay, Incubation, Cell Culture, Expressing, Western Blot

    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)

    Journal: BMC Complementary and Alternative Medicine

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

    doi: 10.1186/s12906-015-0770-9

    Figure Lengend 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)

    Article Snippet: Antibodies against polyadenosine 5′-diphosphate-ribose polymerase (PARP), NFATc1, and β-actin were purchased from Cell Signaling Technology (MA, USA), Santa Cruz Biotechnology (TX, USA), and Sigma Aldrich (MO, USA), respectively.

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

    STAT3-NFATc2 signaling pathway was involved in the SIRT3-mediated fibroblast transdifferention. A-D. Immunoblot analysis of NFATc2 in WT, SIRT3-KO and LV.SIRT3 myofibroblasts. GAPDH expression was used as loading control. The bar graph represents protein quantification measured by densitometry analysis. (n=5). E. Representative immunofluorescence images indicated the subcellular location of NFATc2 in WT and SIRT3-KO myofibroblasts. Nuclei was stained with DAPI. Scale bar: 20 μm. The data are presented as the means ± SEM of three independent experiments. *P

    Journal: American Journal of Translational Research

    Article Title: SIRT3 attenuates AngII-induced cardiac fibrosis by inhibiting myofibroblasts transdifferentiation via STAT3-NFATc2 pathway

    doi:

    Figure Lengend Snippet: STAT3-NFATc2 signaling pathway was involved in the SIRT3-mediated fibroblast transdifferention. A-D. Immunoblot analysis of NFATc2 in WT, SIRT3-KO and LV.SIRT3 myofibroblasts. GAPDH expression was used as loading control. The bar graph represents protein quantification measured by densitometry analysis. (n=5). E. Representative immunofluorescence images indicated the subcellular location of NFATc2 in WT and SIRT3-KO myofibroblasts. Nuclei was stained with DAPI. Scale bar: 20 μm. The data are presented as the means ± SEM of three independent experiments. *P

    Article Snippet: Antibodies against SIRT3 (rabbit monoclonal) (D22A3) [5490], STAT3 (mouse monoclonal) (124H6) [9139], Phospho-Stat3 (Tyr705) (rabbit monoclonal) (D3A7) [9145], NFATc2 (rabbit monoclonal) (D43B1) [5861], Acetylated-Lysine (rabbit polyclonal) [9441] were purchased from Cell Signaling Technology (CST, UK).

    Techniques: Expressing, Immunofluorescence, Staining

    ASIC2 activates the calcineurin/NFAT1 pathway under acidosis. Cells were cultured at pH 7.4 and pH 6.5 for 24 h. The whole-cell extracts and nuclear extracts from (a) SW480 and (b) SW620 cells were used to analyze ASIC2 or NFAT1 by western blotting. Cell invasion ability was measured in the presence or absence of Cyclosporine A at the indicated concentration. c Cyclosporine A significantly inhibited ASIC2-induced invasion of SW480 cells under an acidic environment. d Cyclosporine A significantly inhibited acid-induced invasion of SW620 cells. * p

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: The acid-sensing ion channel, ASIC2, promotes invasion and metastasis of colorectal cancer under acidosis by activating the calcineurin/NFAT1 axis

    doi: 10.1186/s13046-017-0599-9

    Figure Lengend Snippet: ASIC2 activates the calcineurin/NFAT1 pathway under acidosis. Cells were cultured at pH 7.4 and pH 6.5 for 24 h. The whole-cell extracts and nuclear extracts from (a) SW480 and (b) SW620 cells were used to analyze ASIC2 or NFAT1 by western blotting. Cell invasion ability was measured in the presence or absence of Cyclosporine A at the indicated concentration. c Cyclosporine A significantly inhibited ASIC2-induced invasion of SW480 cells under an acidic environment. d Cyclosporine A significantly inhibited acid-induced invasion of SW620 cells. * p

    Article Snippet: ASIC2 was mainly expressed in the cell membrane and cytoplasm (Fig. ), while NFAT1 was expressed in the nucleus and cytoplasm (Additional file : Fig. S5B).

    Techniques: Cell Culture, Western Blot, Concentration Assay

    ChIP-Seq analysis of the NFAT1 binding sites. a ChIP-Seq density heatmap of NFAT1 for all RefSeq genes. The genomic region from -3 kb to +3 kb relative to the TSS is shown. b The top 20 enriched pathways of peak-related genes based on the KEGG or Reactome database

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: The acid-sensing ion channel, ASIC2, promotes invasion and metastasis of colorectal cancer under acidosis by activating the calcineurin/NFAT1 axis

    doi: 10.1186/s13046-017-0599-9

    Figure Lengend Snippet: ChIP-Seq analysis of the NFAT1 binding sites. a ChIP-Seq density heatmap of NFAT1 for all RefSeq genes. The genomic region from -3 kb to +3 kb relative to the TSS is shown. b The top 20 enriched pathways of peak-related genes based on the KEGG or Reactome database

    Article Snippet: ASIC2 was mainly expressed in the cell membrane and cytoplasm (Fig. ), while NFAT1 was expressed in the nucleus and cytoplasm (Additional file : Fig. S5B).

    Techniques: Chromatin Immunoprecipitation, Binding Assay

    Adiponectin-deficient calvarial cells preferentially differentiate into adipocytes. (A–C) Mouse calvarial cells were incubated with β-glycerophosphate (10 mM) and ascorbic acid (50 μM) to induce osteoblast differentiation. (A) The cells were stained for ALP at day 7 and with alizarin red S at day 21 (upper, whole-well image; lower, 40× magnification). (B) Alizarin red S-stained cells were dissolved in 20% methanol and 10% acetic acid and the optical density (OD) was measured at 450 nm. (C) At days 5 and 10, total RNAs were isolated, reverse-transcribed, and subjected to RT-PCR to determine the mRNA levels of BSP, OCN, ALP, β-catenin, Runx2, and β-actin. (D–F) Mouse calvarial cells were incubated with dexamethasone (1 μM), insulin (5 μg/ml), and IBMX (0.5 mM) for the evaluation of adipogenic potential. (D) At day 21, the cells were fixed and stained using oil red O (upper, whole-well image; lower, 40× magnification). (E) The oil red O-stained cells were dissolved in isopropanol and the OD was measured at 500 nm. (F) At day 5, total RNAs were isolated, reverse-transcribed, and subjected to RT-PCR. The ratio of PPARγ to β-actin was obtained by using a densitometer. Data are mean values ± SD of triplicate samples and are representative of three similar independent experiments. * P

    Journal: Frontiers in Endocrinology

    Article Title: Adiponectin Deficiency Triggers Bone Loss by Up-Regulation of Osteoclastogenesis and Down-Regulation of Osteoblastogenesis

    doi: 10.3389/fendo.2019.00815

    Figure Lengend Snippet: Adiponectin-deficient calvarial cells preferentially differentiate into adipocytes. (A–C) Mouse calvarial cells were incubated with β-glycerophosphate (10 mM) and ascorbic acid (50 μM) to induce osteoblast differentiation. (A) The cells were stained for ALP at day 7 and with alizarin red S at day 21 (upper, whole-well image; lower, 40× magnification). (B) Alizarin red S-stained cells were dissolved in 20% methanol and 10% acetic acid and the optical density (OD) was measured at 450 nm. (C) At days 5 and 10, total RNAs were isolated, reverse-transcribed, and subjected to RT-PCR to determine the mRNA levels of BSP, OCN, ALP, β-catenin, Runx2, and β-actin. (D–F) Mouse calvarial cells were incubated with dexamethasone (1 μM), insulin (5 μg/ml), and IBMX (0.5 mM) for the evaluation of adipogenic potential. (D) At day 21, the cells were fixed and stained using oil red O (upper, whole-well image; lower, 40× magnification). (E) The oil red O-stained cells were dissolved in isopropanol and the OD was measured at 500 nm. (F) At day 5, total RNAs were isolated, reverse-transcribed, and subjected to RT-PCR. The ratio of PPARγ to β-actin was obtained by using a densitometer. Data are mean values ± SD of triplicate samples and are representative of three similar independent experiments. * P

    Article Snippet: All other primary antibodies used for immunoblotting were obtained from Cell Signaling Technology (Beverly, MA, USA) except for antibodies against NFATc1, c-Fos (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and β-actin (Sigma-Aldrich).

    Techniques: Incubation, Staining, ALP Assay, Isolation, Reverse Transcription Polymerase Chain Reaction

    Survivin silencing impaired DNA repair by homologous recombination. a qPCR analysis of a set of genes involved in DNA damage repair in Cal51, MDAMB-231, and MCF7 cells depleted or not in Survivin. Data are presented as means (±sem) of ratios normalized to controls from three independent experiments. b HR activity was evaluated by GFP gene conversion assay using the genetically modified RG37 cell line. RG37 cells were first transfected with I-Sce-I coding plasmid, and 24 h later depleted in Survivin or BRCA1 using specific siRNA. After 48 h, % of GFP positive cells was assessed in each condition by flow cytometry and presented as means (±sem) from six independent experiments. c Breast cancer cell lines were depleted in Survivin (SiSurvivin) or not (SiCt) for 48 h and immunoblot analysis were performed to evaluate expression of MUS81/EME1 complex and RAD51 proteins

    Journal: Breast Cancer Research and Treatment

    Article Title: Survivin contributes to DNA repair by homologous recombination in breast cancer cells

    doi: 10.1007/s10549-015-3657-z

    Figure Lengend Snippet: Survivin silencing impaired DNA repair by homologous recombination. a qPCR analysis of a set of genes involved in DNA damage repair in Cal51, MDAMB-231, and MCF7 cells depleted or not in Survivin. Data are presented as means (±sem) of ratios normalized to controls from three independent experiments. b HR activity was evaluated by GFP gene conversion assay using the genetically modified RG37 cell line. RG37 cells were first transfected with I-Sce-I coding plasmid, and 24 h later depleted in Survivin or BRCA1 using specific siRNA. After 48 h, % of GFP positive cells was assessed in each condition by flow cytometry and presented as means (±sem) from six independent experiments. c Breast cancer cell lines were depleted in Survivin (SiSurvivin) or not (SiCt) for 48 h and immunoblot analysis were performed to evaluate expression of MUS81/EME1 complex and RAD51 proteins

    Article Snippet: Survivin antibody was purchased from R & D Systems (Minneapolis, MN, USA), γH2AX from Upstate (Hampshire, UK), Actin from Chemicon International (Billerica, MA, USA), RAD51 from Genetex (Irvine, CA, USA), MUS81 and EME1 from Abcam (Paris, France), p53 from BD Biosciences (Pont de Claix, France), NOXA from Enzo Life Science (Villeurbanne, France), BAX from Dako (Courtaboeuf, France), p21, Ser15 p53, and PUMA from Cell Signaling (Molsheim, France), CyclinB1 from Santa Cruz (Heidelberg, Germany).

    Techniques: Homologous Recombination, Real-time Polymerase Chain Reaction, Activity Assay, Genetically Modified, Transfection, Plasmid Preparation, Flow Cytometry, Cytometry, Expressing

    Gene expression analysis of BIRC5 and HR genes in primary breast tumors. a The correlation map illustrating pairwise correlations among BIRC5, RAD51, EME1, EXO1, BLM , and BRCA1 genes were established using the Breast Cancer Gene-Expression Miner v3.1 web tool. b Kaplan–Meier curves regarding expression of BIRC5, RAD51, EME1, EXO1, BLM , and BRCA1 genes in tumors from patients with breast cancer, were established using the Breast Cancer Gene-Expression Miner v3.1 web tool

    Journal: Breast Cancer Research and Treatment

    Article Title: Survivin contributes to DNA repair by homologous recombination in breast cancer cells

    doi: 10.1007/s10549-015-3657-z

    Figure Lengend Snippet: Gene expression analysis of BIRC5 and HR genes in primary breast tumors. a The correlation map illustrating pairwise correlations among BIRC5, RAD51, EME1, EXO1, BLM , and BRCA1 genes were established using the Breast Cancer Gene-Expression Miner v3.1 web tool. b Kaplan–Meier curves regarding expression of BIRC5, RAD51, EME1, EXO1, BLM , and BRCA1 genes in tumors from patients with breast cancer, were established using the Breast Cancer Gene-Expression Miner v3.1 web tool

    Article Snippet: Survivin antibody was purchased from R & D Systems (Minneapolis, MN, USA), γH2AX from Upstate (Hampshire, UK), Actin from Chemicon International (Billerica, MA, USA), RAD51 from Genetex (Irvine, CA, USA), MUS81 and EME1 from Abcam (Paris, France), p53 from BD Biosciences (Pont de Claix, France), NOXA from Enzo Life Science (Villeurbanne, France), BAX from Dako (Courtaboeuf, France), p21, Ser15 p53, and PUMA from Cell Signaling (Molsheim, France), CyclinB1 from Santa Cruz (Heidelberg, Germany).

    Techniques: Expressing

    NGA inhibited osteoclast gene expression in mouse BMMs in vitro . The expression of mRNAs corresponding to gene products specific to RANKL-induced osteoclast differentiation, including TRAP , CTR , CTSK , and NFATc1 in mouse BMMs treated with 30 ng/ml M-CSF, 50 ng/ml RANKL, and increasing concentrations of NGA (0, 0.2 or 0.4 μg/ml) for 5 days, measured by real-time PCR. RNA expression levels were normalized relative to the expression of GAPDH . Gene expression after being treated with NGA, compared with the 0 μg/ml treatment group (t-test), * p

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

    Article Title: Neogambogic Acid Suppresses Receptor Activator of Nuclear Factor κB Ligand (RANKL)-Induced Osteoclastogenesis by Inhibiting the JNK and NF-κB Pathways in Mouse Bone Marrow-Derived Monocyte/Macrophages

    doi: 10.12659/MSM.909651

    Figure Lengend Snippet: NGA inhibited osteoclast gene expression in mouse BMMs in vitro . The expression of mRNAs corresponding to gene products specific to RANKL-induced osteoclast differentiation, including TRAP , CTR , CTSK , and NFATc1 in mouse BMMs treated with 30 ng/ml M-CSF, 50 ng/ml RANKL, and increasing concentrations of NGA (0, 0.2 or 0.4 μg/ml) for 5 days, measured by real-time PCR. RNA expression levels were normalized relative to the expression of GAPDH . Gene expression after being treated with NGA, compared with the 0 μg/ml treatment group (t-test), * p

    Article Snippet: Specific antibodies against p38, phospho-p38 (Thr180/Tyr182), JNK, phospho-JNK (Thr183/Tyr185), nuclear factor of activated T cells c1 (NFATc1), p65, phospho-p65, GAPDH, and beta-actin were purchased from Cell Signaling Technology (Cambridge, MA, USA).

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

    Madecassoside ( MA ) inhibits the RANKL ‐induced MAPK signaling pathway. (A) Representative western blot images of p‐ JNK 1/2, JNK , p‐ ERK 1/2, p‐p38, p38, ERK , and β‐actin at 0, 10, 20, 30, 60 min stimulated by GST ‐ rRANKL (50 ng/mL) with or without MA (10 μmol L −1 ). (B‐F) The relative ratios of phosphorylated proteins to unphosphorylated proteins were quantitatively determined. The data in the figures represent the means ± SD . Significant differences between the treatment and control groups are indicated as * P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Madecassoside inhibits estrogen deficiency‐induced osteoporosis by suppressing RANKL‐induced osteoclastogenesis, et al. Madecassoside inhibits estrogen deficiency‐induced osteoporosis by suppressing RANKL‐induced osteoclastogenesis

    doi: 10.1111/jcmm.13942

    Figure Lengend Snippet: Madecassoside ( MA ) inhibits the RANKL ‐induced MAPK signaling pathway. (A) Representative western blot images of p‐ JNK 1/2, JNK , p‐ ERK 1/2, p‐p38, p38, ERK , and β‐actin at 0, 10, 20, 30, 60 min stimulated by GST ‐ rRANKL (50 ng/mL) with or without MA (10 μmol L −1 ). (B‐F) The relative ratios of phosphorylated proteins to unphosphorylated proteins were quantitatively determined. The data in the figures represent the means ± SD . Significant differences between the treatment and control groups are indicated as * P

    Article Snippet: Primary antibodies specific for NFATc1, integrin β3, Cathepsin K, V‐ATPase‐d2, IκB‐α, pERK1/2, ERK1/2, and β‐actin were obtained from Santa Cruz Biotechnology (San Jose, CA, USA).

    Techniques: Western Blot

    Madecassoside ( MA ) suppresses NF ‐κB activation and calcium oscillation during osteoclastogenesis. (A) Representative images of western blots reflecting the expression level of IĸB‐α normalized to β‐actin. (B) Quantitative analysis of the fold change in IĸB‐α expression after MA (10 μmol L −1 ) treatment. (C) The bar graph depicts the NF ‐κB luciferase activity of RAW 264.7 cells stably transfected with an NF ‐κB luciferase reporter construct. Cells were treated with varying densities of MA and stimulated by GST ‐ rRANKL (50 ng/mL) for 6 h. (D) Representative images of Ca 2+ oscillation patterns without stimulation by RANKL (M‐ CSF only). (E) Representative images of calcium fluctuation patterns stimulated by RANKL . (F) Representative images of calcium fluctuation patterns stimulated by MA (10 μmol L −1 ) + RANKL . (G) Quantitative analysis of the amplitude of the fluorescence intensity of calcium oscillations in each group. Lines with different colours in each image represent the results of three independent experiments. The data in the figures represent the means ± SD . Significant differences between the treatment and control groups are indicated as * P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Madecassoside inhibits estrogen deficiency‐induced osteoporosis by suppressing RANKL‐induced osteoclastogenesis, et al. Madecassoside inhibits estrogen deficiency‐induced osteoporosis by suppressing RANKL‐induced osteoclastogenesis

    doi: 10.1111/jcmm.13942

    Figure Lengend Snippet: Madecassoside ( MA ) suppresses NF ‐κB activation and calcium oscillation during osteoclastogenesis. (A) Representative images of western blots reflecting the expression level of IĸB‐α normalized to β‐actin. (B) Quantitative analysis of the fold change in IĸB‐α expression after MA (10 μmol L −1 ) treatment. (C) The bar graph depicts the NF ‐κB luciferase activity of RAW 264.7 cells stably transfected with an NF ‐κB luciferase reporter construct. Cells were treated with varying densities of MA and stimulated by GST ‐ rRANKL (50 ng/mL) for 6 h. (D) Representative images of Ca 2+ oscillation patterns without stimulation by RANKL (M‐ CSF only). (E) Representative images of calcium fluctuation patterns stimulated by RANKL . (F) Representative images of calcium fluctuation patterns stimulated by MA (10 μmol L −1 ) + RANKL . (G) Quantitative analysis of the amplitude of the fluorescence intensity of calcium oscillations in each group. Lines with different colours in each image represent the results of three independent experiments. The data in the figures represent the means ± SD . Significant differences between the treatment and control groups are indicated as * P

    Article Snippet: Primary antibodies specific for NFATc1, integrin β3, Cathepsin K, V‐ATPase‐d2, IκB‐α, pERK1/2, ERK1/2, and β‐actin were obtained from Santa Cruz Biotechnology (San Jose, CA, USA).

    Techniques: Activation Assay, Western Blot, Expressing, Luciferase, Activity Assay, Stable Transfection, Transfection, Construct, Fluorescence

    Madecassoside ( MA ) represses NFAT c1 activity and downstream protein expression. (A) The bar graph depicts the NFAT c1 luciferase activity of RAW 264.7 cells stably transfected with an NFAT c1 luciferase reporter construct. Cells were treated with varying densities of MA and stimulated by GST ‐ rRANKL (50 ng/mL) for 24 h. (B)The protein expression of NFAT c1, c‐Fos, V‐ ATP ase‐d2, CTSK , integrin β3 at day 0, day 1, day 3, and day 5 after stimulation by GST ‐ rRANKL (50 ng/mL) with or without MA (10 μmol L −1 ). (C‐G) The statistical significance of differences in protein expression between the MA ‐treated group and control group was analysed. The expression of all the proteins mentioned above was determined relative to β‐actin expression. The data represent the means ± SD . Significant differences between the treatment and control groups are indicated as * P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Madecassoside inhibits estrogen deficiency‐induced osteoporosis by suppressing RANKL‐induced osteoclastogenesis, et al. Madecassoside inhibits estrogen deficiency‐induced osteoporosis by suppressing RANKL‐induced osteoclastogenesis

    doi: 10.1111/jcmm.13942

    Figure Lengend Snippet: Madecassoside ( MA ) represses NFAT c1 activity and downstream protein expression. (A) The bar graph depicts the NFAT c1 luciferase activity of RAW 264.7 cells stably transfected with an NFAT c1 luciferase reporter construct. Cells were treated with varying densities of MA and stimulated by GST ‐ rRANKL (50 ng/mL) for 24 h. (B)The protein expression of NFAT c1, c‐Fos, V‐ ATP ase‐d2, CTSK , integrin β3 at day 0, day 1, day 3, and day 5 after stimulation by GST ‐ rRANKL (50 ng/mL) with or without MA (10 μmol L −1 ). (C‐G) The statistical significance of differences in protein expression between the MA ‐treated group and control group was analysed. The expression of all the proteins mentioned above was determined relative to β‐actin expression. The data represent the means ± SD . Significant differences between the treatment and control groups are indicated as * P

    Article Snippet: Primary antibodies specific for NFATc1, integrin β3, Cathepsin K, V‐ATPase‐d2, IκB‐α, pERK1/2, ERK1/2, and β‐actin were obtained from Santa Cruz Biotechnology (San Jose, CA, USA).

    Techniques: Activity Assay, Expressing, Luciferase, Stable Transfection, Transfection, Construct

    Effect of BBR on HDAC activity and acetylation of histone H3 and α-tubulin. (a) Total cells lysates were incubated with various doses of BBR or 5 μM SAHA in HDAC assay buffer for 3 h. HDAC activity was initiated by adding the HDAC substrate and incubating at 37°C for 1 h. HDAC activity was measured by detecting the OD value at 405 nm. (b) MDA-MB-231 cells were treated with 25 or 50 μM BBR for 24 ~ 72 h or 1 μM SAHA for 24 h. Protein expressions of Ac-histone H3, Ac-α-tubulin and β-actin were analyzed by Western blotting. Images of each indicated probe were cropped from the same blot. (c) MDA-MB-231 cells were treated with 50 μM BBR for 48 h, and nuclear and cytosolic extracts were prepared as described in “Methods”. Protein expressions of Ac-histone H3, histone H3, Ac-α-tubulin, α-tubulin, HDAC1, HDAC2, HDAC6, and GAPDH were analyzed by Western blotting. Images of each indicated probe were cropped from the same blot.

    Journal: Scientific Reports

    Article Title: A gene expression signature-based approach reveals the mechanisms of action of the Chinese herbal medicine berberine

    doi: 10.1038/srep06394

    Figure Lengend Snippet: Effect of BBR on HDAC activity and acetylation of histone H3 and α-tubulin. (a) Total cells lysates were incubated with various doses of BBR or 5 μM SAHA in HDAC assay buffer for 3 h. HDAC activity was initiated by adding the HDAC substrate and incubating at 37°C for 1 h. HDAC activity was measured by detecting the OD value at 405 nm. (b) MDA-MB-231 cells were treated with 25 or 50 μM BBR for 24 ~ 72 h or 1 μM SAHA for 24 h. Protein expressions of Ac-histone H3, Ac-α-tubulin and β-actin were analyzed by Western blotting. Images of each indicated probe were cropped from the same blot. (c) MDA-MB-231 cells were treated with 50 μM BBR for 48 h, and nuclear and cytosolic extracts were prepared as described in “Methods”. Protein expressions of Ac-histone H3, histone H3, Ac-α-tubulin, α-tubulin, HDAC1, HDAC2, HDAC6, and GAPDH were analyzed by Western blotting. Images of each indicated probe were cropped from the same blot.

    Article Snippet: Antibodies specific for β-actin (GTX109639; 1:15000), AKT1 (GTX110613; 1:7500), phospho-Thr308-AKT1 (GTX61798; 1:500), AMPKα1 (GTX112998; 1:5000), phospho-Thr172-AMPKα (GTX63165; 1:2000), ATG5 (GTX113309; 1:2500), eIF2α (GTX101241; 1:250), phospho-Ser51-eIF2α (GTX50300; 1:1500), GAPDH (GTX100118; 1:30000), HDAC1 (GTX100513; 1:5000), HDAC2 (GTX109642; 1:5000), HDAC6 (GTX100722; 1:10000), histone H3 (GTX122148; 1:1000), acetyl-histone H3 (GTX122648; 1:5000), LC3B (GTX127375; 1:10000), mTOR (GTX101558; 1:500), phospho-Ser2448-mTOR (GTX62472; 1:2000), p70S6K (GTX103174; 1:500), phospho-p70S6K (GTX62758; 1:1500), and α-tubulin (GTX112141; 1:5000) were purchased from GeneTex.

    Techniques: Activity Assay, Incubation, Histone Deacetylase Assay, Multiple Displacement Amplification, Western Blot

    rCD200 inhibits RANKL signaling pathway. (A and B) PBMC adherent cells were cultured with M-CSF with or without rCD200 for 48 hr and stimulated for different times with RANKL. Expression of total protein levels and of the phosphorylated forms of p38, ERK, c-fos and NFATc1 was determined by Western blotting. The results of 1 experiment, representative of 3, are presented. (C) rCD200 inhibits osteoclast-related gene expression. Cells were cultivated with M-CSF for 48 hr, RANKL was added on day 3 of culture and medium was changed every 3 days. rCD200 was added at the beginning of the culture and every 3 days. Expression of target gene was determined by real-time PCR after 21 days of culture. Data are mean±SD from 3 independent experiments. *, P

    Journal: PLoS ONE

    Article Title: CD200R/CD200 Inhibits Osteoclastogenesis: New Mechanism of Osteoclast Control by Mesenchymal Stem Cells in Human

    doi: 10.1371/journal.pone.0072831

    Figure Lengend Snippet: rCD200 inhibits RANKL signaling pathway. (A and B) PBMC adherent cells were cultured with M-CSF with or without rCD200 for 48 hr and stimulated for different times with RANKL. Expression of total protein levels and of the phosphorylated forms of p38, ERK, c-fos and NFATc1 was determined by Western blotting. The results of 1 experiment, representative of 3, are presented. (C) rCD200 inhibits osteoclast-related gene expression. Cells were cultivated with M-CSF for 48 hr, RANKL was added on day 3 of culture and medium was changed every 3 days. rCD200 was added at the beginning of the culture and every 3 days. Expression of target gene was determined by real-time PCR after 21 days of culture. Data are mean±SD from 3 independent experiments. *, P

    Article Snippet: The membrane was blocked in 5% milk in PBS/0.1% Tween-20 and probed overnight with the primary antibodies for ERK1/2, phospho-ERK, p38, phospho-p38, c-fos (Cell Signaling Technology, Beverly, CA, USA), NFATc1 (Abcam, Cambridge, MA, USA) and β-actin (Sigma-Aldrich, Lyon, France).

    Techniques: Cell Culture, Expressing, Western Blot, Real-time Polymerase Chain Reaction

    Physical interaction at the NFAT target sequence. DNA oligonucleotide, which includes the NFAT consensus binding sequence GGAAA, was either incubated with the cell lysate of cells stimulated with Ionomycin or untreated cells ( a ) or transiently transfected with ‘Silencer Negative Control’—siRNA or NFATc2-specific siRNA oligonucleotide and also stimulated or not stimulated ( b ). Proteins binding to the biotin-marked oligonucleotide sequence were subsequently precipitated with streptavidin. By means of the respective antibodies, NFATc2 and Sp1 were determined by Western blot analysis

    Journal: BMC Molecular Biology

    Article Title: Interaction between NFATc2 and the transcription factor Sp1 in pancreatic carcinoma cells PaTu 8988t

    doi: 10.1186/s12867-017-0097-9

    Figure Lengend Snippet: Physical interaction at the NFAT target sequence. DNA oligonucleotide, which includes the NFAT consensus binding sequence GGAAA, was either incubated with the cell lysate of cells stimulated with Ionomycin or untreated cells ( a ) or transiently transfected with ‘Silencer Negative Control’—siRNA or NFATc2-specific siRNA oligonucleotide and also stimulated or not stimulated ( b ). Proteins binding to the biotin-marked oligonucleotide sequence were subsequently precipitated with streptavidin. By means of the respective antibodies, NFATc2 and Sp1 were determined by Western blot analysis

    Article Snippet: Similar to NFATc2, 10 min stimulation with Ionomycin led to significant signal amplification of Sp1 protein that decreased after 1 h. As conclusion, Ionomycin may facilitate the interaction between NFATc2 and Sp1 at the early stage of stimulation.

    Techniques: Sequencing, Binding Assay, Incubation, Transfection, Negative Control, Western Blot

    Direct interaction between NFATc2 and Sp1 in GST pull-down assay. Bacteriologically expressed GST and GST-NFATc2 as well as Sp1 previously over-expressed in the pancreatic tumor cells were examined by means of GST pull-down assay; precipitates were determined by Western blot analysis

    Journal: BMC Molecular Biology

    Article Title: Interaction between NFATc2 and the transcription factor Sp1 in pancreatic carcinoma cells PaTu 8988t

    doi: 10.1186/s12867-017-0097-9

    Figure Lengend Snippet: Direct interaction between NFATc2 and Sp1 in GST pull-down assay. Bacteriologically expressed GST and GST-NFATc2 as well as Sp1 previously over-expressed in the pancreatic tumor cells were examined by means of GST pull-down assay; precipitates were determined by Western blot analysis

    Article Snippet: Similar to NFATc2, 10 min stimulation with Ionomycin led to significant signal amplification of Sp1 protein that decreased after 1 h. As conclusion, Ionomycin may facilitate the interaction between NFATc2 and Sp1 at the early stage of stimulation.

    Techniques: Pull Down Assay, Western Blot

    Functional interaction between NFATc2 and Sp1 in luciferase assay. The artificial NFAT-responsive reporter-promotor construct cisNFAT-Luc and the effector plasmids NFATc2 or Sp1, or both, were transiently transfected into the pancreatic tumor cell lines PaTu 8988t, and relative luciferase activity was determined. In the evaluation, the basal activity of the promoter construct cisNFAT-Luc is normalized to the value 100 and the influences of the regulatory changes are indicated in x-fold increase or reduction of this control. The test repeated three times

    Journal: BMC Molecular Biology

    Article Title: Interaction between NFATc2 and the transcription factor Sp1 in pancreatic carcinoma cells PaTu 8988t

    doi: 10.1186/s12867-017-0097-9

    Figure Lengend Snippet: Functional interaction between NFATc2 and Sp1 in luciferase assay. The artificial NFAT-responsive reporter-promotor construct cisNFAT-Luc and the effector plasmids NFATc2 or Sp1, or both, were transiently transfected into the pancreatic tumor cell lines PaTu 8988t, and relative luciferase activity was determined. In the evaluation, the basal activity of the promoter construct cisNFAT-Luc is normalized to the value 100 and the influences of the regulatory changes are indicated in x-fold increase or reduction of this control. The test repeated three times

    Article Snippet: Similar to NFATc2, 10 min stimulation with Ionomycin led to significant signal amplification of Sp1 protein that decreased after 1 h. As conclusion, Ionomycin may facilitate the interaction between NFATc2 and Sp1 at the early stage of stimulation.

    Techniques: Functional Assay, Luciferase, Construct, Transfection, Activity Assay

    NFATc2 becomes translocated into the cell nucleus after stimulation with Ionomycin. Pancreatic tumor cells mixed with serum-free medium for 3 h were stimulated with 0.5 µM of Ionomycin for 0, 10, 30, and 60 min. Evidence is provided by means of NFATc2 antibodies in Western blot analysis ( b ) or by immunofluorescence ( a ). Cell nuclei ( blue ) are stained with DAPI and endogenous NFATc2 ( red ) with alexarot

    Journal: BMC Molecular Biology

    Article Title: Interaction between NFATc2 and the transcription factor Sp1 in pancreatic carcinoma cells PaTu 8988t

    doi: 10.1186/s12867-017-0097-9

    Figure Lengend Snippet: NFATc2 becomes translocated into the cell nucleus after stimulation with Ionomycin. Pancreatic tumor cells mixed with serum-free medium for 3 h were stimulated with 0.5 µM of Ionomycin for 0, 10, 30, and 60 min. Evidence is provided by means of NFATc2 antibodies in Western blot analysis ( b ) or by immunofluorescence ( a ). Cell nuclei ( blue ) are stained with DAPI and endogenous NFATc2 ( red ) with alexarot

    Article Snippet: Similar to NFATc2, 10 min stimulation with Ionomycin led to significant signal amplification of Sp1 protein that decreased after 1 h. As conclusion, Ionomycin may facilitate the interaction between NFATc2 and Sp1 at the early stage of stimulation.

    Techniques: Western Blot, Immunofluorescence, Staining

    Endogenous expression of NFATc2 partner proteins described in the literature and their physical interaction during immunoprecipitation. Proteins were verified in Western blot analysis using the antibodies NFATc2, Sp1, and MEF 2A as well as the loading control ß-actin ( a ). NFATc2 including its interaction partner was subsequently extracted by means of immunoprecipitation ( b )

    Journal: BMC Molecular Biology

    Article Title: Interaction between NFATc2 and the transcription factor Sp1 in pancreatic carcinoma cells PaTu 8988t

    doi: 10.1186/s12867-017-0097-9

    Figure Lengend Snippet: Endogenous expression of NFATc2 partner proteins described in the literature and their physical interaction during immunoprecipitation. Proteins were verified in Western blot analysis using the antibodies NFATc2, Sp1, and MEF 2A as well as the loading control ß-actin ( a ). NFATc2 including its interaction partner was subsequently extracted by means of immunoprecipitation ( b )

    Article Snippet: Similar to NFATc2, 10 min stimulation with Ionomycin led to significant signal amplification of Sp1 protein that decreased after 1 h. As conclusion, Ionomycin may facilitate the interaction between NFATc2 and Sp1 at the early stage of stimulation.

    Techniques: Expressing, Immunoprecipitation, Western Blot

    Co-immunoprecipitation of NFATc2 and Sp1 complexes of pancreatic tumor cells. After 3 h incubation with serum-free medium, the cells were stimulated with 0.5 µM of Ionomycin, harvested, and lysed at defined time points (0, 10, and 60 min). NFATc2 or Sp1 proteins were immunoprecipitated by means of an antibody coupled to an agarose bead. Sp1 binding to NFATc2 ( a ) or NFATc2 binding to Sp1 ( b ) were analyzed by means of Western blotting

    Journal: BMC Molecular Biology

    Article Title: Interaction between NFATc2 and the transcription factor Sp1 in pancreatic carcinoma cells PaTu 8988t

    doi: 10.1186/s12867-017-0097-9

    Figure Lengend Snippet: Co-immunoprecipitation of NFATc2 and Sp1 complexes of pancreatic tumor cells. After 3 h incubation with serum-free medium, the cells were stimulated with 0.5 µM of Ionomycin, harvested, and lysed at defined time points (0, 10, and 60 min). NFATc2 or Sp1 proteins were immunoprecipitated by means of an antibody coupled to an agarose bead. Sp1 binding to NFATc2 ( a ) or NFATc2 binding to Sp1 ( b ) were analyzed by means of Western blotting

    Article Snippet: Similar to NFATc2, 10 min stimulation with Ionomycin led to significant signal amplification of Sp1 protein that decreased after 1 h. As conclusion, Ionomycin may facilitate the interaction between NFATc2 and Sp1 at the early stage of stimulation.

    Techniques: Immunoprecipitation, Incubation, Binding Assay, Western Blot

    Co-localization of NFATc2 and the transcription factor Sp1. PaTu 8988t cells were transiently transfected with NFATc2-GFP. The cells either remained in serum containing medium or, after 24 h, serum was removed for 3 h, and the cells were stimulated with 0.5 µM of Ionomycin for 0, 10, 30, or 60 min. The cells were fixed into paraformaldehyde and incubated with the Sp1 antibody. Cell nuclei ( blue ) are stained with DAPI, endogenous Sp1 ( red ) with alexarot, and NFATc2-GFP green

    Journal: BMC Molecular Biology

    Article Title: Interaction between NFATc2 and the transcription factor Sp1 in pancreatic carcinoma cells PaTu 8988t

    doi: 10.1186/s12867-017-0097-9

    Figure Lengend Snippet: Co-localization of NFATc2 and the transcription factor Sp1. PaTu 8988t cells were transiently transfected with NFATc2-GFP. The cells either remained in serum containing medium or, after 24 h, serum was removed for 3 h, and the cells were stimulated with 0.5 µM of Ionomycin for 0, 10, 30, or 60 min. The cells were fixed into paraformaldehyde and incubated with the Sp1 antibody. Cell nuclei ( blue ) are stained with DAPI, endogenous Sp1 ( red ) with alexarot, and NFATc2-GFP green

    Article Snippet: Similar to NFATc2, 10 min stimulation with Ionomycin led to significant signal amplification of Sp1 protein that decreased after 1 h. As conclusion, Ionomycin may facilitate the interaction between NFATc2 and Sp1 at the early stage of stimulation.

    Techniques: Transfection, Incubation, Staining