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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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Images

1) Product Images from "Gene Expression Profiling of NFATc1-Knockdown in RAW 264.7 Cells: An Alternative Pathway for Macrophage Differentiation"

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

Journal: Cells

doi: 10.3390/cells8020131

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.
Figure Legend 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.

Techniques Used: 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.
Figure Legend 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.

Techniques Used:

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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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.
Figure Legend 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.

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: Expressing

2) Product Images from "Gene Expression Profiling of NFATc1-Knockdown in RAW 264.7 Cells: An Alternative Pathway for Macrophage Differentiation"

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

Journal: Cells

doi: 10.3390/cells8020131

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.
Figure Legend 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.

Techniques Used: 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.
Figure Legend 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.

Techniques Used:

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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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.
Figure Legend 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.

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: Expressing

3) Product Images from "Gene Expression Profiling of NFATc1-Knockdown in RAW 264.7 Cells: An Alternative Pathway for Macrophage Differentiation"

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

Journal: Cells

doi: 10.3390/cells8020131

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.
Figure Legend 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.

Techniques Used: 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.
Figure Legend 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.

Techniques Used:

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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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.
Figure Legend 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.

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: Expressing

4) Product Images from "Gene Expression Profiling of NFATc1-Knockdown in RAW 264.7 Cells: An Alternative Pathway for Macrophage Differentiation"

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

Journal: Cells

doi: 10.3390/cells8020131

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.
Figure Legend 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.

Techniques Used: 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.
Figure Legend 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.

Techniques Used:

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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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.
Figure Legend 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.

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: Expressing

5) Product Images from "Gene Expression Profiling of NFATc1-Knockdown in RAW 264.7 Cells: An Alternative Pathway for Macrophage Differentiation"

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

Journal: Cells

doi: 10.3390/cells8020131

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.
Figure Legend 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.

Techniques Used: 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.
Figure Legend 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.

Techniques Used:

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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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.
Figure Legend 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.

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: Expressing

6) Product Images from "Molecular Signaling Pathways Mediating Osteoclastogenesis Induced by Prostate Cancer Cells"

Article Title: Molecular Signaling Pathways Mediating Osteoclastogenesis Induced by Prostate Cancer Cells

Journal: BMC Cancer

doi: 10.1186/1471-2407-13-605

Prostate cancer-derived factors induce calcium/NFATc1 signaling in osteoclast precursors. A, B) RANKL-primed RAW 264.7 cells were loaded with fura-2-AM, cultured for 15 min untreated ( negative control ), with 50 ng/ml RANKL, or 10% PC3 or LNCaP CM, and imaged for 2 min. A) Average basal calcium levels. Data are means ± SD; n = 32-48 cells per condition; *significance compared to negative control as assessed by Student’s t-test . B) Percentage of active cells classified as having standard deviation of basal calcium above 0.05. C) RANKL-primed bone marrow precursors were pre-treated with 50 μM BAPTA ( white bars ) and exposed to 10% PC3 or LNCaP CM for 2 days, and average number of osteoclasts was assessed. Data are means ± SEM; n = 3-7 experiments; *significance compared to negative control; # significance compared to no inhibitor. D, F) Protein was extracted or samples were fixed from untreated or RANKL-primed RAW 264.7 cells, or RANKL-primed precursors cultured for 2 h untreated, with RANKL, or with 10% PC3 or LNCaP CM. D) NFATc1 protein levels, α-tubulin as a loading control. E) NFATc1 localization ( green ); nuclei were counterstained using DAPI ( blue ). F) Average NFATc1 nuclear localization normalized to the continuous RANKL (RL) treatment. Data are means ± SEM; n = 3 experiments. G, H) RANKL-primed bone marrow cells were pretreated with 0.1% DMSO vehicle ( grey bars ), or 50 μM NFAT inhibitor VIVIT ( white bars ), and cultured untreated, treated with RANKL, or with 10% PC3 or LNCaP CM for 2 h to examine NFATc1 localization; or 2 days to assess osteoclast formation. G) Average percentage of cells with nuclear NFATc1. Data are means ± SEM; n = 3 experiments. H) Average number of osteoclasts. Data are means ± SEM; n = 3-7 experiments; *significance compared to negative control, # significance as compared to no inhibitor.
Figure Legend Snippet: Prostate cancer-derived factors induce calcium/NFATc1 signaling in osteoclast precursors. A, B) RANKL-primed RAW 264.7 cells were loaded with fura-2-AM, cultured for 15 min untreated ( negative control ), with 50 ng/ml RANKL, or 10% PC3 or LNCaP CM, and imaged for 2 min. A) Average basal calcium levels. Data are means ± SD; n = 32-48 cells per condition; *significance compared to negative control as assessed by Student’s t-test . B) Percentage of active cells classified as having standard deviation of basal calcium above 0.05. C) RANKL-primed bone marrow precursors were pre-treated with 50 μM BAPTA ( white bars ) and exposed to 10% PC3 or LNCaP CM for 2 days, and average number of osteoclasts was assessed. Data are means ± SEM; n = 3-7 experiments; *significance compared to negative control; # significance compared to no inhibitor. D, F) Protein was extracted or samples were fixed from untreated or RANKL-primed RAW 264.7 cells, or RANKL-primed precursors cultured for 2 h untreated, with RANKL, or with 10% PC3 or LNCaP CM. D) NFATc1 protein levels, α-tubulin as a loading control. E) NFATc1 localization ( green ); nuclei were counterstained using DAPI ( blue ). F) Average NFATc1 nuclear localization normalized to the continuous RANKL (RL) treatment. Data are means ± SEM; n = 3 experiments. G, H) RANKL-primed bone marrow cells were pretreated with 0.1% DMSO vehicle ( grey bars ), or 50 μM NFAT inhibitor VIVIT ( white bars ), and cultured untreated, treated with RANKL, or with 10% PC3 or LNCaP CM for 2 h to examine NFATc1 localization; or 2 days to assess osteoclast formation. G) Average percentage of cells with nuclear NFATc1. Data are means ± SEM; n = 3 experiments. H) Average number of osteoclasts. Data are means ± SEM; n = 3-7 experiments; *significance compared to negative control, # significance as compared to no inhibitor.

Techniques Used: Derivative Assay, Cell Culture, Negative Control, Standard Deviation

7) Product Images from "Molecular Signaling Pathways Mediating Osteoclastogenesis Induced by Prostate Cancer Cells"

Article Title: Molecular Signaling Pathways Mediating Osteoclastogenesis Induced by Prostate Cancer Cells

Journal: BMC Cancer

doi: 10.1186/1471-2407-13-605

Prostate cancer-derived factors induce calcium/NFATc1 signaling in osteoclast precursors. A, B) RANKL-primed RAW 264.7 cells were loaded with fura-2-AM, cultured for 15 min untreated ( negative control ), with 50 ng/ml RANKL, or 10% PC3 or LNCaP CM, and imaged for 2 min. A) Average basal calcium levels. Data are means ± SD; n = 32-48 cells per condition; *significance compared to negative control as assessed by Student’s t-test . B) Percentage of active cells classified as having standard deviation of basal calcium above 0.05. C) RANKL-primed bone marrow precursors were pre-treated with 50 μM BAPTA ( white bars ) and exposed to 10% PC3 or LNCaP CM for 2 days, and average number of osteoclasts was assessed. Data are means ± SEM; n = 3-7 experiments; *significance compared to negative control; # significance compared to no inhibitor. D, F) Protein was extracted or samples were fixed from untreated or RANKL-primed RAW 264.7 cells, or RANKL-primed precursors cultured for 2 h untreated, with RANKL, or with 10% PC3 or LNCaP CM. D) NFATc1 protein levels, α-tubulin as a loading control. E) NFATc1 localization ( green ); nuclei were counterstained using DAPI ( blue ). F) Average NFATc1 nuclear localization normalized to the continuous RANKL (RL) treatment. Data are means ± SEM; n = 3 experiments. G, H) RANKL-primed bone marrow cells were pretreated with 0.1% DMSO vehicle ( grey bars ), or 50 μM NFAT inhibitor VIVIT ( white bars ), and cultured untreated, treated with RANKL, or with 10% PC3 or LNCaP CM for 2 h to examine NFATc1 localization; or 2 days to assess osteoclast formation. G) Average percentage of cells with nuclear NFATc1. Data are means ± SEM; n = 3 experiments. H) Average number of osteoclasts. Data are means ± SEM; n = 3-7 experiments; *significance compared to negative control, # significance as compared to no inhibitor.
Figure Legend Snippet: Prostate cancer-derived factors induce calcium/NFATc1 signaling in osteoclast precursors. A, B) RANKL-primed RAW 264.7 cells were loaded with fura-2-AM, cultured for 15 min untreated ( negative control ), with 50 ng/ml RANKL, or 10% PC3 or LNCaP CM, and imaged for 2 min. A) Average basal calcium levels. Data are means ± SD; n = 32-48 cells per condition; *significance compared to negative control as assessed by Student’s t-test . B) Percentage of active cells classified as having standard deviation of basal calcium above 0.05. C) RANKL-primed bone marrow precursors were pre-treated with 50 μM BAPTA ( white bars ) and exposed to 10% PC3 or LNCaP CM for 2 days, and average number of osteoclasts was assessed. Data are means ± SEM; n = 3-7 experiments; *significance compared to negative control; # significance compared to no inhibitor. D, F) Protein was extracted or samples were fixed from untreated or RANKL-primed RAW 264.7 cells, or RANKL-primed precursors cultured for 2 h untreated, with RANKL, or with 10% PC3 or LNCaP CM. D) NFATc1 protein levels, α-tubulin as a loading control. E) NFATc1 localization ( green ); nuclei were counterstained using DAPI ( blue ). F) Average NFATc1 nuclear localization normalized to the continuous RANKL (RL) treatment. Data are means ± SEM; n = 3 experiments. G, H) RANKL-primed bone marrow cells were pretreated with 0.1% DMSO vehicle ( grey bars ), or 50 μM NFAT inhibitor VIVIT ( white bars ), and cultured untreated, treated with RANKL, or with 10% PC3 or LNCaP CM for 2 h to examine NFATc1 localization; or 2 days to assess osteoclast formation. G) Average percentage of cells with nuclear NFATc1. Data are means ± SEM; n = 3 experiments. H) Average number of osteoclasts. Data are means ± SEM; n = 3-7 experiments; *significance compared to negative control, # significance as compared to no inhibitor.

Techniques Used: Derivative Assay, Cell Culture, Negative Control, Standard Deviation

8) Product Images from "Effects of salubrinal on development of osteoclasts and osteoblasts from bone marrow-derived cells"

Article Title: Effects of salubrinal on development of osteoclasts and osteoblasts from bone marrow-derived cells

Journal: BMC Musculoskeletal Disorders

doi: 10.1186/1471-2474-14-197

Suppression of RANKL- induced expression of NFATc1 and TRACP by salubrinal in RAW264. 7 cells. A : Salubrinal-driven suppression of NFATc1 expression in RANKL-stimulated bone marrow-derived cells. B : Dose dependent suppression of NFATc1 in response to 1 – 20 μM salubrinal. β-actin was used as a loading control. C : Relative mRNA levels of NFATc1 and TRACP in response to 1 – 20 μM salubrinal. The mRNA levels are normalized by the mRNA level of the sample without salubrinal in the absence of RANKL stimulation.
Figure Legend Snippet: Suppression of RANKL- induced expression of NFATc1 and TRACP by salubrinal in RAW264. 7 cells. A : Salubrinal-driven suppression of NFATc1 expression in RANKL-stimulated bone marrow-derived cells. B : Dose dependent suppression of NFATc1 in response to 1 – 20 μM salubrinal. β-actin was used as a loading control. C : Relative mRNA levels of NFATc1 and TRACP in response to 1 – 20 μM salubrinal. The mRNA levels are normalized by the mRNA level of the sample without salubrinal in the absence of RANKL stimulation.

Techniques Used: Expressing, Derivative Assay

9) Product Images from "Effects of salubrinal on development of osteoclasts and osteoblasts from bone marrow-derived cells"

Article Title: Effects of salubrinal on development of osteoclasts and osteoblasts from bone marrow-derived cells

Journal: BMC Musculoskeletal Disorders

doi: 10.1186/1471-2474-14-197

Suppression of RANKL- induced expression of NFATc1 and TRACP by salubrinal in RAW264. 7 cells. A : Salubrinal-driven suppression of NFATc1 expression in RANKL-stimulated bone marrow-derived cells. B : Dose dependent suppression of NFATc1 in response to 1 – 20 μM salubrinal. β-actin was used as a loading control. C : Relative mRNA levels of NFATc1 and TRACP in response to 1 – 20 μM salubrinal. The mRNA levels are normalized by the mRNA level of the sample without salubrinal in the absence of RANKL stimulation.
Figure Legend Snippet: Suppression of RANKL- induced expression of NFATc1 and TRACP by salubrinal in RAW264. 7 cells. A : Salubrinal-driven suppression of NFATc1 expression in RANKL-stimulated bone marrow-derived cells. B : Dose dependent suppression of NFATc1 in response to 1 – 20 μM salubrinal. β-actin was used as a loading control. C : Relative mRNA levels of NFATc1 and TRACP in response to 1 – 20 μM salubrinal. The mRNA levels are normalized by the mRNA level of the sample without salubrinal in the absence of RANKL stimulation.

Techniques Used: Expressing, Derivative Assay

10) Product Images from "Effects of salubrinal on development of osteoclasts and osteoblasts from bone marrow-derived cells"

Article Title: Effects of salubrinal on development of osteoclasts and osteoblasts from bone marrow-derived cells

Journal: BMC Musculoskeletal Disorders

doi: 10.1186/1471-2474-14-197

Suppression of RANKL- induced expression of NFATc1 and TRACP by salubrinal in RAW264. 7 cells. A : Salubrinal-driven suppression of NFATc1 expression in RANKL-stimulated bone marrow-derived cells. B : Dose dependent suppression of NFATc1 in response to 1 – 20 μM salubrinal. β-actin was used as a loading control. C : Relative mRNA levels of NFATc1 and TRACP in response to 1 – 20 μM salubrinal. The mRNA levels are normalized by the mRNA level of the sample without salubrinal in the absence of RANKL stimulation.
Figure Legend Snippet: Suppression of RANKL- induced expression of NFATc1 and TRACP by salubrinal in RAW264. 7 cells. A : Salubrinal-driven suppression of NFATc1 expression in RANKL-stimulated bone marrow-derived cells. B : Dose dependent suppression of NFATc1 in response to 1 – 20 μM salubrinal. β-actin was used as a loading control. C : Relative mRNA levels of NFATc1 and TRACP in response to 1 – 20 μM salubrinal. The mRNA levels are normalized by the mRNA level of the sample without salubrinal in the absence of RANKL stimulation.

Techniques Used: Expressing, Derivative Assay

11) Product Images from "Amorphigenin inhibits Osteoclast differentiation by suppressing c-Fos and nuclear factor of activated T cells"

Article Title: Amorphigenin inhibits Osteoclast differentiation by suppressing c-Fos and nuclear factor of activated T cells

Journal: Anatomy & Cell Biology

doi: 10.5115/acb.2010.43.4.310

Amorphigenin suppresses RANKL-induced gene expression. BMMs were pretreated with or without amorphigenin and then stimulated with RANKL (100 ng/ml) for the indicated times. Total RNA was isolated from the treated cells. The mRNA expression of c-fos, NFATc1, TRAP, OSCAR, and GAPDH was analyzed by RT-PCR.
Figure Legend Snippet: Amorphigenin suppresses RANKL-induced gene expression. BMMs were pretreated with or without amorphigenin and then stimulated with RANKL (100 ng/ml) for the indicated times. Total RNA was isolated from the treated cells. The mRNA expression of c-fos, NFATc1, TRAP, OSCAR, and GAPDH was analyzed by RT-PCR.

Techniques Used: Expressing, Isolation, Reverse Transcription Polymerase Chain Reaction

Amorphigenin inhibits RANKL-mediated c-Fos and NFATc1 expression. BMMs were pretreated with or without amorphigenin and stimulated with RANKL (100 ng/ml) for the indicated times. The cells were lysed in lysis buffer. c-Fos, NFATc1, or actin proteins were detected by Western blotting with an anti-c-fos, and anti-NFATc1, or anti-actin-antibody, respectively.
Figure Legend Snippet: Amorphigenin inhibits RANKL-mediated c-Fos and NFATc1 expression. BMMs were pretreated with or without amorphigenin and stimulated with RANKL (100 ng/ml) for the indicated times. The cells were lysed in lysis buffer. c-Fos, NFATc1, or actin proteins were detected by Western blotting with an anti-c-fos, and anti-NFATc1, or anti-actin-antibody, respectively.

Techniques Used: Expressing, Lysis, Western Blot

12) Product Images from "Amorphigenin inhibits Osteoclast differentiation by suppressing c-Fos and nuclear factor of activated T cells"

Article Title: Amorphigenin inhibits Osteoclast differentiation by suppressing c-Fos and nuclear factor of activated T cells

Journal: Anatomy & Cell Biology

doi: 10.5115/acb.2010.43.4.310

Amorphigenin suppresses RANKL-induced gene expression. BMMs were pretreated with or without amorphigenin and then stimulated with RANKL (100 ng/ml) for the indicated times. Total RNA was isolated from the treated cells. The mRNA expression of c-fos, NFATc1, TRAP, OSCAR, and GAPDH was analyzed by RT-PCR.
Figure Legend Snippet: Amorphigenin suppresses RANKL-induced gene expression. BMMs were pretreated with or without amorphigenin and then stimulated with RANKL (100 ng/ml) for the indicated times. Total RNA was isolated from the treated cells. The mRNA expression of c-fos, NFATc1, TRAP, OSCAR, and GAPDH was analyzed by RT-PCR.

Techniques Used: Expressing, Isolation, Reverse Transcription Polymerase Chain Reaction

Amorphigenin inhibits RANKL-mediated c-Fos and NFATc1 expression. BMMs were pretreated with or without amorphigenin and stimulated with RANKL (100 ng/ml) for the indicated times. The cells were lysed in lysis buffer. c-Fos, NFATc1, or actin proteins were detected by Western blotting with an anti-c-fos, and anti-NFATc1, or anti-actin-antibody, respectively.
Figure Legend Snippet: Amorphigenin inhibits RANKL-mediated c-Fos and NFATc1 expression. BMMs were pretreated with or without amorphigenin and stimulated with RANKL (100 ng/ml) for the indicated times. The cells were lysed in lysis buffer. c-Fos, NFATc1, or actin proteins were detected by Western blotting with an anti-c-fos, and anti-NFATc1, or anti-actin-antibody, respectively.

Techniques Used: Expressing, Lysis, Western Blot

13) Product Images from "Amorphigenin inhibits Osteoclast differentiation by suppressing c-Fos and nuclear factor of activated T cells"

Article Title: Amorphigenin inhibits Osteoclast differentiation by suppressing c-Fos and nuclear factor of activated T cells

Journal: Anatomy & Cell Biology

doi: 10.5115/acb.2010.43.4.310

Amorphigenin suppresses RANKL-induced gene expression. BMMs were pretreated with or without amorphigenin and then stimulated with RANKL (100 ng/ml) for the indicated times. Total RNA was isolated from the treated cells. The mRNA expression of c-fos, NFATc1, TRAP, OSCAR, and GAPDH was analyzed by RT-PCR.
Figure Legend Snippet: Amorphigenin suppresses RANKL-induced gene expression. BMMs were pretreated with or without amorphigenin and then stimulated with RANKL (100 ng/ml) for the indicated times. Total RNA was isolated from the treated cells. The mRNA expression of c-fos, NFATc1, TRAP, OSCAR, and GAPDH was analyzed by RT-PCR.

Techniques Used: Expressing, Isolation, Reverse Transcription Polymerase Chain Reaction

Amorphigenin inhibits RANKL-mediated c-Fos and NFATc1 expression. BMMs were pretreated with or without amorphigenin and stimulated with RANKL (100 ng/ml) for the indicated times. The cells were lysed in lysis buffer. c-Fos, NFATc1, or actin proteins were detected by Western blotting with an anti-c-fos, and anti-NFATc1, or anti-actin-antibody, respectively.
Figure Legend Snippet: Amorphigenin inhibits RANKL-mediated c-Fos and NFATc1 expression. BMMs were pretreated with or without amorphigenin and stimulated with RANKL (100 ng/ml) for the indicated times. The cells were lysed in lysis buffer. c-Fos, NFATc1, or actin proteins were detected by Western blotting with an anti-c-fos, and anti-NFATc1, or anti-actin-antibody, respectively.

Techniques Used: Expressing, Lysis, Western Blot

14) Product Images from "Effect of Cornus Officinalis on Receptor Activator of Nuclear Factor-kappaB Ligand (RANKL)-induced Osteoclast Differentiation"

Article Title: Effect of Cornus Officinalis on Receptor Activator of Nuclear Factor-kappaB Ligand (RANKL)-induced Osteoclast Differentiation

Journal: Journal of Bone Metabolism

doi: 10.11005/jbm.2012.19.2.121

Cornus officinalis inhibits the expression of c-Fos and nuclear factor of activated T cells cytoplasmic 1 (NFATc1). Bone marrow-derived macrophages (BMMs) were pretreated with or without Cornus officinalis (100 µg/mL) and further stimulated with receptor activator of nuclear factor-kappaB ligand (RANKL; 100 n/mL) for the indicated time. The cells were lysed and proteins were revolved by 10% SDS-polyacrylamide gel electrophoresis (PAGE) and subjected to Western blot analysis. Similar results were obtained in three independent experiments. DMSO, dimethyl sulfoxide
Figure Legend Snippet: Cornus officinalis inhibits the expression of c-Fos and nuclear factor of activated T cells cytoplasmic 1 (NFATc1). Bone marrow-derived macrophages (BMMs) were pretreated with or without Cornus officinalis (100 µg/mL) and further stimulated with receptor activator of nuclear factor-kappaB ligand (RANKL; 100 n/mL) for the indicated time. The cells were lysed and proteins were revolved by 10% SDS-polyacrylamide gel electrophoresis (PAGE) and subjected to Western blot analysis. Similar results were obtained in three independent experiments. DMSO, dimethyl sulfoxide

Techniques Used: Expressing, Derivative Assay, Polyacrylamide Gel Electrophoresis, Western Blot

Cornus officinalis inhibits osteoclast-specific gene expression. bone marrow-derived macrophages (BMMs) were treated with or without Cornus officinalis (100 µg/mL) and further stimulated with receptor activator of nuclear factor-kappaB ligand (RANKL; 100 n/mL) for the indicated time. cDNA was synthesized by using total RNA and reverse transcriptase-polymerase chain reaction (RT-PCR) was performed using cDNA. PCR products were separated by electrophoresis in an agarose gel and visualized with ethidium bromide staining. Similar results were obtained in three independent experiments. DMSO, dimethyl sulfoxide; TRAP, tartrate-resistant acid phosphatase; OSCAR, osteoclast-associated receptor; NFATc1, nuclear factor of activated T cells cytoplasmic 1, GAPDH, glyceraldehyde-3-phosphate dehydrogenase
Figure Legend Snippet: Cornus officinalis inhibits osteoclast-specific gene expression. bone marrow-derived macrophages (BMMs) were treated with or without Cornus officinalis (100 µg/mL) and further stimulated with receptor activator of nuclear factor-kappaB ligand (RANKL; 100 n/mL) for the indicated time. cDNA was synthesized by using total RNA and reverse transcriptase-polymerase chain reaction (RT-PCR) was performed using cDNA. PCR products were separated by electrophoresis in an agarose gel and visualized with ethidium bromide staining. Similar results were obtained in three independent experiments. DMSO, dimethyl sulfoxide; TRAP, tartrate-resistant acid phosphatase; OSCAR, osteoclast-associated receptor; NFATc1, nuclear factor of activated T cells cytoplasmic 1, GAPDH, glyceraldehyde-3-phosphate dehydrogenase

Techniques Used: Expressing, Derivative Assay, Synthesized, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Electrophoresis, Agarose Gel Electrophoresis, Staining

15) Product Images from "Effect of Cornus Officinalis on Receptor Activator of Nuclear Factor-kappaB Ligand (RANKL)-induced Osteoclast Differentiation"

Article Title: Effect of Cornus Officinalis on Receptor Activator of Nuclear Factor-kappaB Ligand (RANKL)-induced Osteoclast Differentiation

Journal: Journal of Bone Metabolism

doi: 10.11005/jbm.2012.19.2.121

Cornus officinalis inhibits the expression of c-Fos and nuclear factor of activated T cells cytoplasmic 1 (NFATc1). Bone marrow-derived macrophages (BMMs) were pretreated with or without Cornus officinalis (100 µg/mL) and further stimulated with receptor activator of nuclear factor-kappaB ligand (RANKL; 100 n/mL) for the indicated time. The cells were lysed and proteins were revolved by 10% SDS-polyacrylamide gel electrophoresis (PAGE) and subjected to Western blot analysis. Similar results were obtained in three independent experiments. DMSO, dimethyl sulfoxide
Figure Legend Snippet: Cornus officinalis inhibits the expression of c-Fos and nuclear factor of activated T cells cytoplasmic 1 (NFATc1). Bone marrow-derived macrophages (BMMs) were pretreated with or without Cornus officinalis (100 µg/mL) and further stimulated with receptor activator of nuclear factor-kappaB ligand (RANKL; 100 n/mL) for the indicated time. The cells were lysed and proteins were revolved by 10% SDS-polyacrylamide gel electrophoresis (PAGE) and subjected to Western blot analysis. Similar results were obtained in three independent experiments. DMSO, dimethyl sulfoxide

Techniques Used: Expressing, Derivative Assay, Polyacrylamide Gel Electrophoresis, Western Blot

Cornus officinalis inhibits osteoclast-specific gene expression. bone marrow-derived macrophages (BMMs) were treated with or without Cornus officinalis (100 µg/mL) and further stimulated with receptor activator of nuclear factor-kappaB ligand (RANKL; 100 n/mL) for the indicated time. cDNA was synthesized by using total RNA and reverse transcriptase-polymerase chain reaction (RT-PCR) was performed using cDNA. PCR products were separated by electrophoresis in an agarose gel and visualized with ethidium bromide staining. Similar results were obtained in three independent experiments. DMSO, dimethyl sulfoxide; TRAP, tartrate-resistant acid phosphatase; OSCAR, osteoclast-associated receptor; NFATc1, nuclear factor of activated T cells cytoplasmic 1, GAPDH, glyceraldehyde-3-phosphate dehydrogenase
Figure Legend Snippet: Cornus officinalis inhibits osteoclast-specific gene expression. bone marrow-derived macrophages (BMMs) were treated with or without Cornus officinalis (100 µg/mL) and further stimulated with receptor activator of nuclear factor-kappaB ligand (RANKL; 100 n/mL) for the indicated time. cDNA was synthesized by using total RNA and reverse transcriptase-polymerase chain reaction (RT-PCR) was performed using cDNA. PCR products were separated by electrophoresis in an agarose gel and visualized with ethidium bromide staining. Similar results were obtained in three independent experiments. DMSO, dimethyl sulfoxide; TRAP, tartrate-resistant acid phosphatase; OSCAR, osteoclast-associated receptor; NFATc1, nuclear factor of activated T cells cytoplasmic 1, GAPDH, glyceraldehyde-3-phosphate dehydrogenase

Techniques Used: Expressing, Derivative Assay, Synthesized, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Electrophoresis, Agarose Gel Electrophoresis, Staining

16) Product Images from "Bu-Shen-Ning-Xin decoction: inhibition of osteoclastogenesis by abrogation of the RANKL-induced NFATc1 and NF-κB signaling pathways via selective estrogen receptor α"

Article Title: Bu-Shen-Ning-Xin decoction: inhibition of osteoclastogenesis by abrogation of the RANKL-induced NFATc1 and NF-κB signaling pathways via selective estrogen receptor α

Journal: Drug Design, Development and Therapy

doi: 10.2147/DDDT.S88512

Summary of the inhibitory effects of BSNXD on osteoclast differentiation. Notes: The suppression of BSNXD on RANKL-induced activation of the NFATc1 via ERα pathway is correlated with the inhibition of osteoclastogenesis. The attenuation of the ERα-dependent NF-κB signaling pathway may correspond to a mechanism that contributes to the suppression of NFATc1 expression by BSNXD. Abbreviations: BSNXD, Bu-Shen-Ning-Xin decoction; RANKL, receptor activation of nuclear factor κB ligand; ER, estrogen receptor; NFATc1, nuclear factor of activated T-cells, cytoplasmic 1 1; M-CSF, macrophage-colony-stimulating factor.
Figure Legend Snippet: Summary of the inhibitory effects of BSNXD on osteoclast differentiation. Notes: The suppression of BSNXD on RANKL-induced activation of the NFATc1 via ERα pathway is correlated with the inhibition of osteoclastogenesis. The attenuation of the ERα-dependent NF-κB signaling pathway may correspond to a mechanism that contributes to the suppression of NFATc1 expression by BSNXD. Abbreviations: BSNXD, Bu-Shen-Ning-Xin decoction; RANKL, receptor activation of nuclear factor κB ligand; ER, estrogen receptor; NFATc1, nuclear factor of activated T-cells, cytoplasmic 1 1; M-CSF, macrophage-colony-stimulating factor.

Techniques Used: Activation Assay, Inhibition, Expressing

BSNXD inhibits RANKL-induced NFATc1 accumulation via inhibition of the NF-κB signaling pathway in osteoclast precursor cells through ERα. Notes: ( A ) Treatment with the 20% OVX + BSNXD mid-dose group-derived serum significantly decreased NF-κB-luciferase activity compared with treatment with 20% control serum; pretreatment with the selective ERα antagonist (MPP) but not the ERβ antagonist (R,R-THC) or AR antagonist flutamide (FLUT) blocked the effect induced by the BSNXD-derived serum. ( B ) The ratio of phospho-PLCγ2/total PLCγ2 remained similar during osteoclastogenesis in the different groups. ( C ) Treatment with 20% OVX + BSNXD mid-dose group-derived serum inhibited RANKL-induced NFATc1 expression in osteoclast precursor cells. This effect involved ERα but not ERβ or AR. “+” represents containing; “−” represents not containing. Data are expressed as means ± SEM (n=10). ** P
Figure Legend Snippet: BSNXD inhibits RANKL-induced NFATc1 accumulation via inhibition of the NF-κB signaling pathway in osteoclast precursor cells through ERα. Notes: ( A ) Treatment with the 20% OVX + BSNXD mid-dose group-derived serum significantly decreased NF-κB-luciferase activity compared with treatment with 20% control serum; pretreatment with the selective ERα antagonist (MPP) but not the ERβ antagonist (R,R-THC) or AR antagonist flutamide (FLUT) blocked the effect induced by the BSNXD-derived serum. ( B ) The ratio of phospho-PLCγ2/total PLCγ2 remained similar during osteoclastogenesis in the different groups. ( C ) Treatment with 20% OVX + BSNXD mid-dose group-derived serum inhibited RANKL-induced NFATc1 expression in osteoclast precursor cells. This effect involved ERα but not ERβ or AR. “+” represents containing; “−” represents not containing. Data are expressed as means ± SEM (n=10). ** P

Techniques Used: Inhibition, Derivative Assay, Luciferase, Activity Assay, Expressing

17) Product Images from "Water extract of Magnolia officinalis cortex inhibits osteoclastogenesis and bone resorption by downregulation of nuclear factor of activated T cells cytoplasmic 1"

Article Title: Water extract of Magnolia officinalis cortex inhibits osteoclastogenesis and bone resorption by downregulation of nuclear factor of activated T cells cytoplasmic 1

Journal: Integrative Medicine Research

doi: 10.1016/j.imr.2015.02.002

Effect of WEMC on RANKL-induced c-Fos and NFATc1 expression in BMMs. BMMs were pretreated with vehicle or WEMC (80 μg/mL) for 3 hours, and then further cultured with RANKL (100 ng/mL). Total cell lysate or RNA was obtained at the indicated time points. (A) Total cell lysates (30 μg) were subjected to Western blot analysis with the indicated antibodies. β-actin was used as a loading control. (B) mRNA expression levels of c-Fos, NFATc1, and cathepsin K were analyzed by quantitative polymerase chain reaction. (C) BMMs infected with pMX-GFP (control retrovirus) or retrovirus encoding constitutively active NFATc1 were cultured with vehicle or WEMC (80 μg/mL) in the presence of M-CSF (60 ng/mL) and RANKL (100 ng/mL) for 4 days. The number of osteoclasts was counted. BMM, bone marrow macrophage; M-CSF, macrophage colony-stimulating factor; NFATc-1, nuclear factor of activated T cells cytoplasmic 1; RANKL, receptor activator of nuclear factor-κB ligand; WEMC, water extract of Magnolia officinalis cortex.
Figure Legend Snippet: Effect of WEMC on RANKL-induced c-Fos and NFATc1 expression in BMMs. BMMs were pretreated with vehicle or WEMC (80 μg/mL) for 3 hours, and then further cultured with RANKL (100 ng/mL). Total cell lysate or RNA was obtained at the indicated time points. (A) Total cell lysates (30 μg) were subjected to Western blot analysis with the indicated antibodies. β-actin was used as a loading control. (B) mRNA expression levels of c-Fos, NFATc1, and cathepsin K were analyzed by quantitative polymerase chain reaction. (C) BMMs infected with pMX-GFP (control retrovirus) or retrovirus encoding constitutively active NFATc1 were cultured with vehicle or WEMC (80 μg/mL) in the presence of M-CSF (60 ng/mL) and RANKL (100 ng/mL) for 4 days. The number of osteoclasts was counted. BMM, bone marrow macrophage; M-CSF, macrophage colony-stimulating factor; NFATc-1, nuclear factor of activated T cells cytoplasmic 1; RANKL, receptor activator of nuclear factor-κB ligand; WEMC, water extract of Magnolia officinalis cortex.

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

18) Product Images from "TNFα Increases RANKL Expression via PGE2-Induced Activation of NFATc1"

Article Title: TNFα Increases RANKL Expression via PGE2-Induced Activation of NFATc1

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms18030495

The PGE 2 receptor antagonists AH6809 and AH23848 suppress the TNFα-induced activation of NFAT and binding of NFATc1 and CREB to the RANKL promoter in C2C12 cells. ( A ) PGE 2 receptor antagonists significantly inhibited TNFα induction of RANKL only after incubation for 12 and 24 h; ( B ) AH6809 and AH23848 blocked TNFα-mediated RANKL , NFATc1, and CREB expression. AH6809 and AH23848 partially suppressed TNFα-induced NFATc1 protein expression. C2C12 cells were incubated with AH6809 (5 μM) or AH23848 (5 μM) in the presence or absence of TNFα for 24 h; ( C ) PGE 2 receptor antagonists abolished TNFα-induced NFAT transcriptional activity; ( D ) PGE 2 receptor antagonists prevented TNFα-induced NFAT and CREB binding to the mouse RANKL promoter (* p
Figure Legend Snippet: The PGE 2 receptor antagonists AH6809 and AH23848 suppress the TNFα-induced activation of NFAT and binding of NFATc1 and CREB to the RANKL promoter in C2C12 cells. ( A ) PGE 2 receptor antagonists significantly inhibited TNFα induction of RANKL only after incubation for 12 and 24 h; ( B ) AH6809 and AH23848 blocked TNFα-mediated RANKL , NFATc1, and CREB expression. AH6809 and AH23848 partially suppressed TNFα-induced NFATc1 protein expression. C2C12 cells were incubated with AH6809 (5 μM) or AH23848 (5 μM) in the presence or absence of TNFα for 24 h; ( C ) PGE 2 receptor antagonists abolished TNFα-induced NFAT transcriptional activity; ( D ) PGE 2 receptor antagonists prevented TNFα-induced NFAT and CREB binding to the mouse RANKL promoter (* p

Techniques Used: Activation Assay, Binding Assay, Incubation, Expressing, Activity Assay

The COX inhibitor indomethacin blocks TNFα-induced binding of NFATc1 and cAMP response element-binding protein (CREB) to the RANKL promoter in C2C12 cells. ( A ) PGE 2 increased the expression of RANKL , NFATc1, CREB, and COX2, whereas indomethacin suppressed TNFα-mediated RANKL , NFATc1, CREB, and COX2 expression. Treatment with PGE 2 in the presence of indomethacin and TNFα partially rescued the expression of RANKL , NFATc1, CREB, and COX2. C2C12 cells were incubated with the indicated reagents for 24 h and subjected to RT-PCR and western blot analyses; ( B ) PGE 2 increased RANKL promoter–reporter activity in a CREB binding element-dependent manner. C2C12 cells were transfected with RANKL -WT-luc or a CREB-binding site mutant ( RANKL -MT(C)-luc) RANKL promoter, incubated for 24 h in the presence of PGE 2 , and subjected to a luciferase assay; ( C ) PGE 2 induced CREB binding to the mouse RANKL promoter. C2C12 cells were incubated for 24 h with PGE 2 and subjected to a ChIP assay with CREB and control IgG antibodies. The RANKL promoter region containing the CREB-binding element was then amplified; ( D ) Indomethacin prevented TNFα-induced NFAT and CREB binding to the mouse RANKL promoter. C2C12 cells were incubated for 24 h with the indicated reagents and subjected to a ChIP assay (* p
Figure Legend Snippet: The COX inhibitor indomethacin blocks TNFα-induced binding of NFATc1 and cAMP response element-binding protein (CREB) to the RANKL promoter in C2C12 cells. ( A ) PGE 2 increased the expression of RANKL , NFATc1, CREB, and COX2, whereas indomethacin suppressed TNFα-mediated RANKL , NFATc1, CREB, and COX2 expression. Treatment with PGE 2 in the presence of indomethacin and TNFα partially rescued the expression of RANKL , NFATc1, CREB, and COX2. C2C12 cells were incubated with the indicated reagents for 24 h and subjected to RT-PCR and western blot analyses; ( B ) PGE 2 increased RANKL promoter–reporter activity in a CREB binding element-dependent manner. C2C12 cells were transfected with RANKL -WT-luc or a CREB-binding site mutant ( RANKL -MT(C)-luc) RANKL promoter, incubated for 24 h in the presence of PGE 2 , and subjected to a luciferase assay; ( C ) PGE 2 induced CREB binding to the mouse RANKL promoter. C2C12 cells were incubated for 24 h with PGE 2 and subjected to a ChIP assay with CREB and control IgG antibodies. The RANKL promoter region containing the CREB-binding element was then amplified; ( D ) Indomethacin prevented TNFα-induced NFAT and CREB binding to the mouse RANKL promoter. C2C12 cells were incubated for 24 h with the indicated reagents and subjected to a ChIP assay (* p

Techniques Used: Binding Assay, Expressing, Incubation, Reverse Transcription Polymerase Chain Reaction, Western Blot, Activity Assay, Transfection, Mutagenesis, Luciferase, Chromatin Immunoprecipitation, Amplification

Calcineurin/nuclear factor of activated T-cells (NFAT) activation is involved in tumor necrosis factor α (TNFα)-induced receptor activator of nuclear factor-κB ligand ( RANKL ) expression in C2C12 cells. ( A ) TNFα increased RANKL expression in a time-dependent manner. C2C12 cells were incubated in the presence of 10 ng/mL TNFα for the indicated time periods and subjected to quantitative reverse transcription-polymerase chain reaction (RT-PCR) and western blot analyses. Quantitative data are presented as means ± standard deviations (SD); ( B ) TNFα induces NFAT transcriptional activity. C2C12 cells were transfected with a reporter plasmid containing an NFAT response element, exposed to TNFα for 24 h, and subjected to a luciferase assay. Data are presented as firefly luciferase activity levels relative to Renilla activity; ( C ) Inhibition of the calcineurin/NFAT pathway blocked TNFα-mediated RANKL expression. C2C12 cells pretreated with FK506 (10 μg/mL) or cyclosporin A (10 μg/mL) were treated with TNFα for 24 h and subjected to RT-PCR and western blot analyses; ( D ) TNFα increases NFAT binding to the mouse RANKL promoter. C2C12 cells were incubated for 24 h with TNFα, after which a chromatin immunoprecipitation (ChIP) assay was performed using an antibody against NFATc1, with IgG serving as a negative control. The RANKL promoter region containing the NFAT binding element was amplified via PCR. Quantitative ChIP data were normalized to the input and are presented as values relative to vehicle-treated control samples (CON) ( E ) TNFα increased RANKL promoter–reporter activity in an NFAT binding element-dependent manner. C2C12 cells were transfected with a wild-type ( RANKL -WT-luc) or NFAT-binding site mutant ( RANKL -MT(N)-luc) RANKL promoter reporter, incubated for 24 h in the presence of TNFα or NFAT overexpression vector, and subjected to a luciferase assay (* p
Figure Legend Snippet: Calcineurin/nuclear factor of activated T-cells (NFAT) activation is involved in tumor necrosis factor α (TNFα)-induced receptor activator of nuclear factor-κB ligand ( RANKL ) expression in C2C12 cells. ( A ) TNFα increased RANKL expression in a time-dependent manner. C2C12 cells were incubated in the presence of 10 ng/mL TNFα for the indicated time periods and subjected to quantitative reverse transcription-polymerase chain reaction (RT-PCR) and western blot analyses. Quantitative data are presented as means ± standard deviations (SD); ( B ) TNFα induces NFAT transcriptional activity. C2C12 cells were transfected with a reporter plasmid containing an NFAT response element, exposed to TNFα for 24 h, and subjected to a luciferase assay. Data are presented as firefly luciferase activity levels relative to Renilla activity; ( C ) Inhibition of the calcineurin/NFAT pathway blocked TNFα-mediated RANKL expression. C2C12 cells pretreated with FK506 (10 μg/mL) or cyclosporin A (10 μg/mL) were treated with TNFα for 24 h and subjected to RT-PCR and western blot analyses; ( D ) TNFα increases NFAT binding to the mouse RANKL promoter. C2C12 cells were incubated for 24 h with TNFα, after which a chromatin immunoprecipitation (ChIP) assay was performed using an antibody against NFATc1, with IgG serving as a negative control. The RANKL promoter region containing the NFAT binding element was amplified via PCR. Quantitative ChIP data were normalized to the input and are presented as values relative to vehicle-treated control samples (CON) ( E ) TNFα increased RANKL promoter–reporter activity in an NFAT binding element-dependent manner. C2C12 cells were transfected with a wild-type ( RANKL -WT-luc) or NFAT-binding site mutant ( RANKL -MT(N)-luc) RANKL promoter reporter, incubated for 24 h in the presence of TNFα or NFAT overexpression vector, and subjected to a luciferase assay (* p

Techniques Used: Activation Assay, Expressing, Incubation, Reverse Transcription Polymerase Chain Reaction, Western Blot, Activity Assay, Transfection, Plasmid Preparation, Luciferase, Inhibition, Binding Assay, Chromatin Immunoprecipitation, Negative Control, Amplification, Polymerase Chain Reaction, Mutagenesis, Over Expression

PGE 2 production and NFAT activation are necessary for TNFα-induced RANKL expression in primary cultured mouse calvarial cells. ( A ) TNFα enhanced the expression of RANKL and COX2. Calvarial cells were incubated in the presence of TNFα for the indicated time periods and subjected to RT-PCR and western blot analyses; ( B ) ChIP assays revealed that TNFα increased NFATc1 and CREB binding to their cognate binding sites in the mouse RANKL promoter; ( C ) A calcineurin/NFAT signaling inhibitor suppressed TNFα-induced RANKL expression. Calvarial cells were incubated for 24 h in the presence of FK506 and TNFα; ( D ) Treatment with a COX2 inhibitor or PGE 2 receptor antagonists suppressed TNFα-induced RANKL expression. (* p
Figure Legend Snippet: PGE 2 production and NFAT activation are necessary for TNFα-induced RANKL expression in primary cultured mouse calvarial cells. ( A ) TNFα enhanced the expression of RANKL and COX2. Calvarial cells were incubated in the presence of TNFα for the indicated time periods and subjected to RT-PCR and western blot analyses; ( B ) ChIP assays revealed that TNFα increased NFATc1 and CREB binding to their cognate binding sites in the mouse RANKL promoter; ( C ) A calcineurin/NFAT signaling inhibitor suppressed TNFα-induced RANKL expression. Calvarial cells were incubated for 24 h in the presence of FK506 and TNFα; ( D ) Treatment with a COX2 inhibitor or PGE 2 receptor antagonists suppressed TNFα-induced RANKL expression. (* p

Techniques Used: Activation Assay, Expressing, Cell Culture, Incubation, Reverse Transcription Polymerase Chain Reaction, Western Blot, Chromatin Immunoprecipitation, Binding Assay

19) Product Images from "Poligoni Multiflori Radix enhances osteoblast formation and reduces osteoclast differentiation"

Article Title: Poligoni Multiflori Radix enhances osteoblast formation and reduces osteoclast differentiation

Journal: International Journal of Molecular Medicine

doi: 10.3892/ijmm.2018.3603

Effects of PMR on the RANKL-induced expression of c-Fos and NFATc1 in BMMs. BMMs were treated with PMR and then stimulated with M-CSF (30 ng/ml) and RANKL (100 ng/ml) at the indicated time periods and concentrations. (A) The mRNA expression levels of c-Fos and (B) NFATc1 were analyzed by reverse transcription-quantitative polymerase chain reaction. (C) Western blot analysis was performed to confirm the results at the protein level. β-actin was used as an internal control. ** P
Figure Legend Snippet: Effects of PMR on the RANKL-induced expression of c-Fos and NFATc1 in BMMs. BMMs were treated with PMR and then stimulated with M-CSF (30 ng/ml) and RANKL (100 ng/ml) at the indicated time periods and concentrations. (A) The mRNA expression levels of c-Fos and (B) NFATc1 were analyzed by reverse transcription-quantitative polymerase chain reaction. (C) Western blot analysis was performed to confirm the results at the protein level. β-actin was used as an internal control. ** P

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

20) Product Images from "MicroRNA-100-5p inhibits osteoclastogenesis and bone resorption by regulating fibroblast growth factor 21"

Article Title: MicroRNA-100-5p inhibits osteoclastogenesis and bone resorption by regulating fibroblast growth factor 21

Journal: International Journal of Molecular Medicine

doi: 10.3892/ijmm.2018.4017

Agomir-miR regulates osteoblast- and osteoclast-specific gene expression in OVX-operated mice. (A) Mice in the OVX group were injected intravenously with agomir-miR-100-5p or agomir-Con, and the expression levels of miR-100-5p were measured by RT-qPCR in the tibia. (B) Osteoblast-specific genes, Runx2, osterix, osteocalcin and ALP, were detected in the tibia by RT-qPCR. (C) Ratio of RANKL mRNA to OPG mRNA in the tibia was calculated as a marker of osteoclast activity. (D) RT-qPCR was performed to analyze the mRNA expression levels of osteoclast-specific genes in the tibia. (E and F) Protein expression levels of RANK, RANKL and NFATc1 in the tibia were detected using western blotting. Triplicate experiments were performed in each group. * P
Figure Legend Snippet: Agomir-miR regulates osteoblast- and osteoclast-specific gene expression in OVX-operated mice. (A) Mice in the OVX group were injected intravenously with agomir-miR-100-5p or agomir-Con, and the expression levels of miR-100-5p were measured by RT-qPCR in the tibia. (B) Osteoblast-specific genes, Runx2, osterix, osteocalcin and ALP, were detected in the tibia by RT-qPCR. (C) Ratio of RANKL mRNA to OPG mRNA in the tibia was calculated as a marker of osteoclast activity. (D) RT-qPCR was performed to analyze the mRNA expression levels of osteoclast-specific genes in the tibia. (E and F) Protein expression levels of RANK, RANKL and NFATc1 in the tibia were detected using western blotting. Triplicate experiments were performed in each group. * P

Techniques Used: Expressing, Mouse Assay, Injection, Quantitative RT-PCR, ALP Assay, Marker, Activity Assay, Western Blot

Schematic diagram of how miR-100-5p may mediate the suppression of osteoclastogenesis by targeting the hepatokine FGF-21. CA2, carbonic anhydrase 2; Ctsk, cathepsin K; FGF21, fibroblast growth factor 21; miR, microRNA; MMP-9, matrix metalloproteinase-9; NFATc1, nuclear factor of activated T cells, cytoplasmic 1; RANK, receptor activator of nuclear factor-κB; RANKL, RANK ligand.
Figure Legend Snippet: Schematic diagram of how miR-100-5p may mediate the suppression of osteoclastogenesis by targeting the hepatokine FGF-21. CA2, carbonic anhydrase 2; Ctsk, cathepsin K; FGF21, fibroblast growth factor 21; miR, microRNA; MMP-9, matrix metalloproteinase-9; NFATc1, nuclear factor of activated T cells, cytoplasmic 1; RANK, receptor activator of nuclear factor-κB; RANKL, RANK ligand.

Techniques Used:

21) Product Images from "Pod1/Tcf21 is regulated by retinoic acid signaling and inhibits differentiation of epicardium-derived cells into smooth muscle in the developing heart"

Article Title: Pod1/Tcf21 is regulated by retinoic acid signaling and inhibits differentiation of epicardium-derived cells into smooth muscle in the developing heart

Journal: Developmental biology

doi: 10.1016/j.ydbio.2012.06.002

RA activates Pod1 and WT1 , but not NFATC1 or Tbx18 , gene expression in cultured chick proepicardial (PE) cells and EPDCs
Figure Legend Snippet: RA activates Pod1 and WT1 , but not NFATC1 or Tbx18 , gene expression in cultured chick proepicardial (PE) cells and EPDCs

Techniques Used: Expressing, Cell Culture

Pod1, WT1, NFATC1, and Tbx18 are heterogeneously expressed in epicardium and EPDCs of chick E7 and mouse E14.5 hearts
Figure Legend Snippet: Pod1, WT1, NFATC1, and Tbx18 are heterogeneously expressed in epicardium and EPDCs of chick E7 and mouse E14.5 hearts

Techniques Used:

22) Product Images from "The Dual Role of Oat Bran Water Extract in Bone Homeostasis Through the Regulation of Osteoclastogenesis and Osteoblast Differentiation"

Article Title: The Dual Role of Oat Bran Water Extract in Bone Homeostasis Through the Regulation of Osteoclastogenesis and Osteoblast Differentiation

Journal: Molecules

doi: 10.3390/molecules23123119

OBWE inhibits the RANKL-induced expression level of c-Fos/NFATc1 through the alteration of I-κB. ( A ) The BMMs were stimulated with RANKL (10 ng/mL) and M-CSF (30 ng/mL) in the presence or absence of OBWE (10 µg/mL) for the indicated times. Total RNA was then isolated using TRIzol reagent and the mRNA expression levels were evaluated using real-time PCR. GAPDH was used as the internal control. * p
Figure Legend Snippet: OBWE inhibits the RANKL-induced expression level of c-Fos/NFATc1 through the alteration of I-κB. ( A ) The BMMs were stimulated with RANKL (10 ng/mL) and M-CSF (30 ng/mL) in the presence or absence of OBWE (10 µg/mL) for the indicated times. Total RNA was then isolated using TRIzol reagent and the mRNA expression levels were evaluated using real-time PCR. GAPDH was used as the internal control. * p

Techniques Used: Expressing, Isolation, Real-time Polymerase Chain Reaction

23) Product Images from "The E3 ubiquitin ligase GRAIL regulates T cell tolerance and regulatory T cell function by mediating T cell receptor-CD3 degradation"

Article Title: The E3 ubiquitin ligase GRAIL regulates T cell tolerance and regulatory T cell function by mediating T cell receptor-CD3 degradation

Journal: Immunity

doi: 10.1016/j.immuni.2010.05.002

GRAIL negatively regulates NFATc1 expression in activated naïve and regulatory T cells (A and B) mRNA expression of indicated genes in flow cytometry-sorted Foxp3-GFP + CD4 + nTreg from WT and Rnf128 −/− mice activated with anti-CD3 for 6 hours in the absence or presence of CsA, anti-IL-2 and anti-IL-21 antibodies were analyzed by real-time RT-PCR. The graph shows means ± standard deviation (SD). The results shown are a representative of at least two independent experiments. (C and D) mRNA expression of various genes in naïve CD4 + T cells from WT or Rnf128 −/− mice activated with anti-CD3 for 24 hours in the absence or presence of CsA, anti-IL-2 and anti-IL-21 antibodies was analyzed by real-time RT-PCR. The graph shows means ± standard deviation (SD). The results shown are a representative of at least two independent experiments.
Figure Legend Snippet: GRAIL negatively regulates NFATc1 expression in activated naïve and regulatory T cells (A and B) mRNA expression of indicated genes in flow cytometry-sorted Foxp3-GFP + CD4 + nTreg from WT and Rnf128 −/− mice activated with anti-CD3 for 6 hours in the absence or presence of CsA, anti-IL-2 and anti-IL-21 antibodies were analyzed by real-time RT-PCR. The graph shows means ± standard deviation (SD). The results shown are a representative of at least two independent experiments. (C and D) mRNA expression of various genes in naïve CD4 + T cells from WT or Rnf128 −/− mice activated with anti-CD3 for 24 hours in the absence or presence of CsA, anti-IL-2 and anti-IL-21 antibodies was analyzed by real-time RT-PCR. The graph shows means ± standard deviation (SD). The results shown are a representative of at least two independent experiments.

Techniques Used: Expressing, Flow Cytometry, Cytometry, Mouse Assay, Quantitative RT-PCR, Standard Deviation

24) Product Images from "The calcineurin-NFAT pathway negatively regulates megakaryopoiesis"

Article Title: The calcineurin-NFAT pathway negatively regulates megakaryopoiesis

Journal: Blood

doi: 10.1182/blood-2012-04-421172

Increased expression of DSCR1 attenuates calcineurin signaling and leads to an increase in megakaryocytes in vivo. (A) Bone marrow (BM) cytospins from wild-type (WT) and Dscr1 Tg mice were treated with and without ionophore and immunostained with anti-NFATc1
Figure Legend Snippet: Increased expression of DSCR1 attenuates calcineurin signaling and leads to an increase in megakaryocytes in vivo. (A) Bone marrow (BM) cytospins from wild-type (WT) and Dscr1 Tg mice were treated with and without ionophore and immunostained with anti-NFATc1

Techniques Used: Expressing, In Vivo, Mouse Assay

CsA stimulates megakaryopoiesis in vivo. (A) Bone marrow cytospins from wild-type mice treated with the calcineurin inhibitor CsA or vehicle for 6 weeks were immunostained for NFATc1 (red) and Dapi (blue) to examine steady-state NFATc1 subcellular localization.
Figure Legend Snippet: CsA stimulates megakaryopoiesis in vivo. (A) Bone marrow cytospins from wild-type mice treated with the calcineurin inhibitor CsA or vehicle for 6 weeks were immunostained for NFATc1 (red) and Dapi (blue) to examine steady-state NFATc1 subcellular localization.

Techniques Used: In Vivo, Mouse Assay

25) Product Images from "Flavonoids compounds from Tridax procumbens inhibit osteoclast differentiation by down‐regulating c‐Fos activation. Flavonoids compounds from Tridax procumbens inhibit osteoclast differentiation by down‐regulating c‐Fos activation"

Article Title: Flavonoids compounds from Tridax procumbens inhibit osteoclast differentiation by down‐regulating c‐Fos activation. Flavonoids compounds from Tridax procumbens inhibit osteoclast differentiation by down‐regulating c‐Fos activation

Journal: Journal of Cellular and Molecular Medicine

doi: 10.1111/jcmm.14948

The effect of TPF on LPS‐induced osteoclast‐specific transcription factors. (A, B) c‐Fos protein synthesis, (C) expression of c‐Fos gene, (D, E) NFATc1 protein synthesis, (F) expression of NFATc1 gene, (G, H) Ap‐1 protein synthesis and (I) expression of AP‐1 gene. The data were represented as mean ± SD (n = 5) of 5 independent experiments. * P
Figure Legend Snippet: The effect of TPF on LPS‐induced osteoclast‐specific transcription factors. (A, B) c‐Fos protein synthesis, (C) expression of c‐Fos gene, (D, E) NFATc1 protein synthesis, (F) expression of NFATc1 gene, (G, H) Ap‐1 protein synthesis and (I) expression of AP‐1 gene. The data were represented as mean ± SD (n = 5) of 5 independent experiments. * P

Techniques Used: Expressing

26) Product Images from "Adenosine A1 receptor regulates osteoclast formation by altering TRAF6/TAK1 signaling"

Article Title: Adenosine A1 receptor regulates osteoclast formation by altering TRAF6/TAK1 signaling

Journal: Purinergic Signalling

doi: 10.1007/s11302-012-9292-9

A proposed scheme of adenosine/A 1 R-mediated regulation of RANKL-induced osteoclast formation. During osteoclastogenesis, the critical transcription factors c-fos and NFATc1 are induced by RANKL. NFATc1 is known to be master switch of osteoclastogenesis.
Figure Legend Snippet: A proposed scheme of adenosine/A 1 R-mediated regulation of RANKL-induced osteoclast formation. During osteoclastogenesis, the critical transcription factors c-fos and NFATc1 are induced by RANKL. NFATc1 is known to be master switch of osteoclastogenesis.

Techniques Used:

Suppression of the RANKL-induced expression of transcription factors NFATc1 and c-fos by A 1 R-selective antagonist. BMMs were cultured with M-CSF and RANKL (30 ng/ml each), with or without various concentrations of DPCPX for 4 days ( a ,
Figure Legend Snippet: Suppression of the RANKL-induced expression of transcription factors NFATc1 and c-fos by A 1 R-selective antagonist. BMMs were cultured with M-CSF and RANKL (30 ng/ml each), with or without various concentrations of DPCPX for 4 days ( a ,

Techniques Used: Expressing, Cell Culture

27) Product Images from "Notch Suppresses Nuclear Factor of Activated T Cells (Nfat) Transactivation and Nfatc1 Expression in Chondrocytes"

Article Title: Notch Suppresses Nuclear Factor of Activated T Cells (Nfat) Transactivation and Nfatc1 Expression in Chondrocytes

Journal: Endocrinology

doi: 10.1210/en.2012-1925

Notch inhibits Nfat transactivation and Nfatc1 expression in chondrocytes. Primary Rosa Notch chondrocytes were infected with Ad-CMV-Cre (NICD, black bars) or with Ad-CMV-GFP (GFP, white bars) as control. A, Subconfluent cells were transfected with an
Figure Legend Snippet: Notch inhibits Nfat transactivation and Nfatc1 expression in chondrocytes. Primary Rosa Notch chondrocytes were infected with Ad-CMV-Cre (NICD, black bars) or with Ad-CMV-GFP (GFP, white bars) as control. A, Subconfluent cells were transfected with an

Techniques Used: Expressing, Infection, Transfection

Notch and Nfatc1 regulate chondrocyte differentiation. Primary Rosa Notch chondrocytes were infected with Ad-CMV-caNfatc1 (Nfatc1, black bars) or with Ad-CMV-GFP (GFP, white bars) as control, either in the context of NICD overexpression (NICD) or under
Figure Legend Snippet: Notch and Nfatc1 regulate chondrocyte differentiation. Primary Rosa Notch chondrocytes were infected with Ad-CMV-caNfatc1 (Nfatc1, black bars) or with Ad-CMV-GFP (GFP, white bars) as control, either in the context of NICD overexpression (NICD) or under

Techniques Used: Infection, Over Expression

28) Product Images from "Mechanical Loading Regulates NFATc1 and ?-Catenin Signaling through a GSK3? Control Node *"

Article Title: Mechanical Loading Regulates NFATc1 and ?-Catenin Signaling through a GSK3? Control Node *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M109.039453

Mechanical strain promotes NFATc1 nuclear accumulation and increases in COX2 by inhibition of GSK3β. A , mechanical strain was applied to cells for the indicated time. Nuclear NFATc1 bands are shown in the top row , with PARP to verify nuclear origin
Figure Legend Snippet: Mechanical strain promotes NFATc1 nuclear accumulation and increases in COX2 by inhibition of GSK3β. A , mechanical strain was applied to cells for the indicated time. Nuclear NFATc1 bands are shown in the top row , with PARP to verify nuclear origin

Techniques Used: Inhibition

Inhibition of calcium/calcineurin signaling does not block strain induced NFATc1 nuclear accumulation. A , cells were treated with ionomycin (1 μ m ) the for indicated times, and proteins from nuclear and cytoplasmic fractions were immunoblotted
Figure Legend Snippet: Inhibition of calcium/calcineurin signaling does not block strain induced NFATc1 nuclear accumulation. A , cells were treated with ionomycin (1 μ m ) the for indicated times, and proteins from nuclear and cytoplasmic fractions were immunoblotted

Techniques Used: Inhibition, Blocking Assay

NFATc1 is critical to mechanical stimulation of COX2, but not for mechanical repression of adipogenesis. A , NFATc1 was targeted with siRNA and cell proteins analyzed 4 h after strain application. In the presence of NFATc1 knockdown, COX2 did not respond
Figure Legend Snippet: NFATc1 is critical to mechanical stimulation of COX2, but not for mechanical repression of adipogenesis. A , NFATc1 was targeted with siRNA and cell proteins analyzed 4 h after strain application. In the presence of NFATc1 knockdown, COX2 did not respond

Techniques Used:

29) Product Images from "Mechanical Loading Regulates NFATc1 and ?-Catenin Signaling through a GSK3? Control Node *"

Article Title: Mechanical Loading Regulates NFATc1 and ?-Catenin Signaling through a GSK3? Control Node *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M109.039453

Mechanical strain promotes NFATc1 nuclear accumulation and increases in COX2 by inhibition of GSK3β. A , mechanical strain was applied to cells for the indicated time. Nuclear NFATc1 bands are shown in the top row , with PARP to verify nuclear origin
Figure Legend Snippet: Mechanical strain promotes NFATc1 nuclear accumulation and increases in COX2 by inhibition of GSK3β. A , mechanical strain was applied to cells for the indicated time. Nuclear NFATc1 bands are shown in the top row , with PARP to verify nuclear origin

Techniques Used: Inhibition

Inhibition of calcium/calcineurin signaling does not block strain induced NFATc1 nuclear accumulation. A , cells were treated with ionomycin (1 μ m ) the for indicated times, and proteins from nuclear and cytoplasmic fractions were immunoblotted
Figure Legend Snippet: Inhibition of calcium/calcineurin signaling does not block strain induced NFATc1 nuclear accumulation. A , cells were treated with ionomycin (1 μ m ) the for indicated times, and proteins from nuclear and cytoplasmic fractions were immunoblotted

Techniques Used: Inhibition, Blocking Assay

NFATc1 is critical to mechanical stimulation of COX2, but not for mechanical repression of adipogenesis. A , NFATc1 was targeted with siRNA and cell proteins analyzed 4 h after strain application. In the presence of NFATc1 knockdown, COX2 did not respond
Figure Legend Snippet: NFATc1 is critical to mechanical stimulation of COX2, but not for mechanical repression of adipogenesis. A , NFATc1 was targeted with siRNA and cell proteins analyzed 4 h after strain application. In the presence of NFATc1 knockdown, COX2 did not respond

Techniques Used:

30) Product Images from "Mechanical Loading Regulates NFATc1 and ?-Catenin Signaling through a GSK3? Control Node *"

Article Title: Mechanical Loading Regulates NFATc1 and ?-Catenin Signaling through a GSK3? Control Node *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M109.039453

Mechanical strain promotes NFATc1 nuclear accumulation and increases in COX2 by inhibition of GSK3β. A , mechanical strain was applied to cells for the indicated time. Nuclear NFATc1 bands are shown in the top row , with PARP to verify nuclear origin
Figure Legend Snippet: Mechanical strain promotes NFATc1 nuclear accumulation and increases in COX2 by inhibition of GSK3β. A , mechanical strain was applied to cells for the indicated time. Nuclear NFATc1 bands are shown in the top row , with PARP to verify nuclear origin

Techniques Used: Inhibition

Inhibition of calcium/calcineurin signaling does not block strain induced NFATc1 nuclear accumulation. A , cells were treated with ionomycin (1 μ m ) the for indicated times, and proteins from nuclear and cytoplasmic fractions were immunoblotted
Figure Legend Snippet: Inhibition of calcium/calcineurin signaling does not block strain induced NFATc1 nuclear accumulation. A , cells were treated with ionomycin (1 μ m ) the for indicated times, and proteins from nuclear and cytoplasmic fractions were immunoblotted

Techniques Used: Inhibition, Blocking Assay

NFATc1 is critical to mechanical stimulation of COX2, but not for mechanical repression of adipogenesis. A , NFATc1 was targeted with siRNA and cell proteins analyzed 4 h after strain application. In the presence of NFATc1 knockdown, COX2 did not respond
Figure Legend Snippet: NFATc1 is critical to mechanical stimulation of COX2, but not for mechanical repression of adipogenesis. A , NFATc1 was targeted with siRNA and cell proteins analyzed 4 h after strain application. In the presence of NFATc1 knockdown, COX2 did not respond

Techniques Used:

31) Product Images from "3BP2 Adapter Protein Is Required for Receptor Activator of NF?B Ligand (RANKL)-induced Osteoclast Differentiation of RAW264.7 Cells *"

Article Title: 3BP2 Adapter Protein Is Required for Receptor Activator of NF?B Ligand (RANKL)-induced Osteoclast Differentiation of RAW264.7 Cells *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M109.091124

Complementation of osteoclast formation by ectopic expression of constitutively active NFATc1 in 3BP2 knockdown cells. shLacZ and sh3BP2 cells were transfected with control vector or pRV-HA-caNFAT2-IRES-GFP vector encoding HA-tagged constitutively active
Figure Legend Snippet: Complementation of osteoclast formation by ectopic expression of constitutively active NFATc1 in 3BP2 knockdown cells. shLacZ and sh3BP2 cells were transfected with control vector or pRV-HA-caNFAT2-IRES-GFP vector encoding HA-tagged constitutively active

Techniques Used: Expressing, Transfection, Plasmid Preparation

32) Product Images from "Tmem178 acts in a novel negative feedback loop targeting NFATc1 to regulate bone mass"

Article Title: Tmem178 acts in a novel negative feedback loop targeting NFATc1 to regulate bone mass

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.1511285112

( A ) Tmem178 expression in a gene array comparing WT and PLCγ2 −/− BMMs in suspension (Susp) or plated on pRGD for 4 h (Adh). ( B ) Quantification of osteoblast numbers per bone perimeter [N.Ob./ B.Pm (mm −1 ) in WT and Tmem178 −/− mice]. n = 4 per genotype. n.s., nonsignificant. ( C ) Quantification of mineral apposition rate (MAR) of 4-wk-old WT and Tmem178 −/− mice labeled with calcein and alizarin red. n = 5 per genotype. ( D ) Quantification of bone formation rate (BFR) of mice in C . ( E ) Representative images of dynamic histomorphometry quantified in C and D . ( F ) Q-RT PCR of RANKL mRNA in whole long bones devoid of marrow cells from WT or Tmem178 −/− mice. n = 4 per genotype. ( G ) Q-RT PCR of OPG mRNA from mice in F . ( H ) Q-RT PCR of Tmem178 mRNA in WT BMMs, OBs, and OCs cultured in vitro. Expression levels were normalized to cyclophilin. ( I ) Expression of Tmem178 mRNA during RANKL-induced osteoclastogenesis in vitro in control (NFATc1 fl/fl ) and NFATc1-deficient cells (NFATc1 Δ/Δ ). * P
Figure Legend Snippet: ( A ) Tmem178 expression in a gene array comparing WT and PLCγ2 −/− BMMs in suspension (Susp) or plated on pRGD for 4 h (Adh). ( B ) Quantification of osteoblast numbers per bone perimeter [N.Ob./ B.Pm (mm −1 ) in WT and Tmem178 −/− mice]. n = 4 per genotype. n.s., nonsignificant. ( C ) Quantification of mineral apposition rate (MAR) of 4-wk-old WT and Tmem178 −/− mice labeled with calcein and alizarin red. n = 5 per genotype. ( D ) Quantification of bone formation rate (BFR) of mice in C . ( E ) Representative images of dynamic histomorphometry quantified in C and D . ( F ) Q-RT PCR of RANKL mRNA in whole long bones devoid of marrow cells from WT or Tmem178 −/− mice. n = 4 per genotype. ( G ) Q-RT PCR of OPG mRNA from mice in F . ( H ) Q-RT PCR of Tmem178 mRNA in WT BMMs, OBs, and OCs cultured in vitro. Expression levels were normalized to cyclophilin. ( I ) Expression of Tmem178 mRNA during RANKL-induced osteoclastogenesis in vitro in control (NFATc1 fl/fl ) and NFATc1-deficient cells (NFATc1 Δ/Δ ). * P

Techniques Used: Expressing, Mouse Assay, Labeling, Reverse Transcription Polymerase Chain Reaction, Cell Culture, In Vitro

( A ) Representative immunofluorescence images of NFATc1 (red) in WT and Tmem178 −/− BMMs cultured with 10 ng/mL M-CSF and 50 ng/mL RANKL for the indicated days. DAPI (blue) was used as nuclear stain. ( B ) Western blot analysis of NFATc1 nuclear translocation in response to 50 ng/mL RANKL in WT and Tmem178 −/− preOCs for the indicated time points. Lamin B is shown as loading control, representative of three experiments. ( C ) Western blot analysis of NFATc1 nuclear translocation in response to 50 ng/mL RANKL in WT cells expressing empty vector pMX or Tmem178-HA for the indicated time points. Histone 3 is shown as loading control, representative of three experiments.
Figure Legend Snippet: ( A ) Representative immunofluorescence images of NFATc1 (red) in WT and Tmem178 −/− BMMs cultured with 10 ng/mL M-CSF and 50 ng/mL RANKL for the indicated days. DAPI (blue) was used as nuclear stain. ( B ) Western blot analysis of NFATc1 nuclear translocation in response to 50 ng/mL RANKL in WT and Tmem178 −/− preOCs for the indicated time points. Lamin B is shown as loading control, representative of three experiments. ( C ) Western blot analysis of NFATc1 nuclear translocation in response to 50 ng/mL RANKL in WT cells expressing empty vector pMX or Tmem178-HA for the indicated time points. Histone 3 is shown as loading control, representative of three experiments.

Techniques Used: Immunofluorescence, Cell Culture, Staining, Western Blot, Translocation Assay, Expressing, Plasmid Preparation

Tmem178 controls RANKL-induced NFATc1 activation. ( A ) Q-RT PCR analysis of NFATc1 during osteoclastogenesis. ** P
Figure Legend Snippet: Tmem178 controls RANKL-induced NFATc1 activation. ( A ) Q-RT PCR analysis of NFATc1 during osteoclastogenesis. ** P

Techniques Used: Activation Assay, Reverse Transcription Polymerase Chain Reaction

33) Product Images from "NFATc1 regulates lymphatic endothelial development"

Article Title: NFATc1 regulates lymphatic endothelial development

Journal: Mechanisms of development

doi: 10.1016/j.mod.2009.02.003

Activation of VEGFR-2 induces NFATc1 nuclear localization in LEC in vitro
Figure Legend Snippet: Activation of VEGFR-2 induces NFATc1 nuclear localization in LEC in vitro

Techniques Used: Activation Assay, In Vitro

NFATc1 activates LEC specific gene promoters
Figure Legend Snippet: NFATc1 activates LEC specific gene promoters

Techniques Used:

NFATc1 regulates VEGF-A induced LEC gene expression
Figure Legend Snippet: NFATc1 regulates VEGF-A induced LEC gene expression

Techniques Used: Expressing

A model for the role of NFATc1 in lymphatic lineage selection in development and injury
Figure Legend Snippet: A model for the role of NFATc1 in lymphatic lineage selection in development and injury

Techniques Used: Selection

Diminished NFATc1 signaling is associated with dysregulated lymphatic patterning
Figure Legend Snippet: Diminished NFATc1 signaling is associated with dysregulated lymphatic patterning

Techniques Used:

In utero cyclosporine-A treatment inhibits NFATc1 nuclear localization, diminishes lymphatic podoplanin and FGFR-3 expression and leads to irregular lymphatic patterning from the jugular lymph sacs
Figure Legend Snippet: In utero cyclosporine-A treatment inhibits NFATc1 nuclear localization, diminishes lymphatic podoplanin and FGFR-3 expression and leads to irregular lymphatic patterning from the jugular lymph sacs

Techniques Used: In Utero, Expressing

NFATc1 regulates endogenous expression of LEC genes
Figure Legend Snippet: NFATc1 regulates endogenous expression of LEC genes

Techniques Used: Expressing

NFATc1 is coexpressed with Prox-1 and VEGFR-3 on developing and mature lymphatic endothelial cells
Figure Legend Snippet: NFATc1 is coexpressed with Prox-1 and VEGFR-3 on developing and mature lymphatic endothelial cells

Techniques Used:

34) Product Images from "NFATc1 regulates lymphatic endothelial development"

Article Title: NFATc1 regulates lymphatic endothelial development

Journal: Mechanisms of development

doi: 10.1016/j.mod.2009.02.003

Activation of VEGFR-2 induces NFATc1 nuclear localization in LEC in vitro
Figure Legend Snippet: Activation of VEGFR-2 induces NFATc1 nuclear localization in LEC in vitro

Techniques Used: Activation Assay, In Vitro

NFATc1 activates LEC specific gene promoters
Figure Legend Snippet: NFATc1 activates LEC specific gene promoters

Techniques Used:

NFATc1 regulates VEGF-A induced LEC gene expression
Figure Legend Snippet: NFATc1 regulates VEGF-A induced LEC gene expression

Techniques Used: Expressing

A model for the role of NFATc1 in lymphatic lineage selection in development and injury
Figure Legend Snippet: A model for the role of NFATc1 in lymphatic lineage selection in development and injury

Techniques Used: Selection

Diminished NFATc1 signaling is associated with dysregulated lymphatic patterning
Figure Legend Snippet: Diminished NFATc1 signaling is associated with dysregulated lymphatic patterning

Techniques Used:

In utero cyclosporine-A treatment inhibits NFATc1 nuclear localization, diminishes lymphatic podoplanin and FGFR-3 expression and leads to irregular lymphatic patterning from the jugular lymph sacs
Figure Legend Snippet: In utero cyclosporine-A treatment inhibits NFATc1 nuclear localization, diminishes lymphatic podoplanin and FGFR-3 expression and leads to irregular lymphatic patterning from the jugular lymph sacs

Techniques Used: In Utero, Expressing

NFATc1 regulates endogenous expression of LEC genes
Figure Legend Snippet: NFATc1 regulates endogenous expression of LEC genes

Techniques Used: Expressing

NFATc1 is coexpressed with Prox-1 and VEGFR-3 on developing and mature lymphatic endothelial cells
Figure Legend Snippet: NFATc1 is coexpressed with Prox-1 and VEGFR-3 on developing and mature lymphatic endothelial cells

Techniques Used:

35) Product Images from "Segregated Foxc2, NFATc1 and Connexin expression at normal developing venous valves, and Connexin-specific differences in the valve phenotypes of Cx37, Cx43, and Cx47 knockout mice"

Article Title: Segregated Foxc2, NFATc1 and Connexin expression at normal developing venous valves, and Connexin-specific differences in the valve phenotypes of Cx37, Cx43, and Cx47 knockout mice

Journal: Developmental biology

doi: 10.1016/j.ydbio.2016.02.033

Segregated Foxc2 and NFATc1 expression in developing peripheral VVs correlates closely with polarized Cx expression. Sections of P7-P11 VVs were triple immunostained for Foxc2/Cx37/Cx43 (A); NFATc1/Cx47/Cx37 (D); NFATc1/Cx43/Cx37 (E); Foxc2/NFATc1/Cx37
Figure Legend Snippet: Segregated Foxc2 and NFATc1 expression in developing peripheral VVs correlates closely with polarized Cx expression. Sections of P7-P11 VVs were triple immunostained for Foxc2/Cx37/Cx43 (A); NFATc1/Cx47/Cx37 (D); NFATc1/Cx43/Cx37 (E); Foxc2/NFATc1/Cx37

Techniques Used: Expressing

36) Product Images from "Segregated Foxc2, NFATc1 and Connexin expression at normal developing venous valves, and Connexin-specific differences in the valve phenotypes of Cx37, Cx43, and Cx47 knockout mice"

Article Title: Segregated Foxc2, NFATc1 and Connexin expression at normal developing venous valves, and Connexin-specific differences in the valve phenotypes of Cx37, Cx43, and Cx47 knockout mice

Journal: Developmental biology

doi: 10.1016/j.ydbio.2016.02.033

Segregated Foxc2 and NFATc1 expression in developing peripheral VVs correlates closely with polarized Cx expression. Sections of P7-P11 VVs were triple immunostained for Foxc2/Cx37/Cx43 (A); NFATc1/Cx47/Cx37 (D); NFATc1/Cx43/Cx37 (E); Foxc2/NFATc1/Cx37
Figure Legend Snippet: Segregated Foxc2 and NFATc1 expression in developing peripheral VVs correlates closely with polarized Cx expression. Sections of P7-P11 VVs were triple immunostained for Foxc2/Cx37/Cx43 (A); NFATc1/Cx47/Cx37 (D); NFATc1/Cx43/Cx37 (E); Foxc2/NFATc1/Cx37

Techniques Used: Expressing

37) Product Images from "Segregated Foxc2, NFATc1 and Connexin expression at normal developing venous valves, and Connexin-specific differences in the valve phenotypes of Cx37, Cx43, and Cx47 knockout mice"

Article Title: Segregated Foxc2, NFATc1 and Connexin expression at normal developing venous valves, and Connexin-specific differences in the valve phenotypes of Cx37, Cx43, and Cx47 knockout mice

Journal: Developmental biology

doi: 10.1016/j.ydbio.2016.02.033

Segregated Foxc2 and NFATc1 expression in developing peripheral VVs correlates closely with polarized Cx expression. Sections of P7-P11 VVs were triple immunostained for Foxc2/Cx37/Cx43 (A); NFATc1/Cx47/Cx37 (D); NFATc1/Cx43/Cx37 (E); Foxc2/NFATc1/Cx37
Figure Legend Snippet: Segregated Foxc2 and NFATc1 expression in developing peripheral VVs correlates closely with polarized Cx expression. Sections of P7-P11 VVs were triple immunostained for Foxc2/Cx37/Cx43 (A); NFATc1/Cx47/Cx37 (D); NFATc1/Cx43/Cx37 (E); Foxc2/NFATc1/Cx37

Techniques Used: Expressing

38) Product Images from "Segregated Foxc2, NFATc1 and Connexin expression at normal developing venous valves, and Connexin-specific differences in the valve phenotypes of Cx37, Cx43, and Cx47 knockout mice"

Article Title: Segregated Foxc2, NFATc1 and Connexin expression at normal developing venous valves, and Connexin-specific differences in the valve phenotypes of Cx37, Cx43, and Cx47 knockout mice

Journal: Developmental biology

doi: 10.1016/j.ydbio.2016.02.033

Segregated Foxc2 and NFATc1 expression in developing peripheral VVs correlates closely with polarized Cx expression. Sections of P7-P11 VVs were triple immunostained for Foxc2/Cx37/Cx43 (A); NFATc1/Cx47/Cx37 (D); NFATc1/Cx43/Cx37 (E); Foxc2/NFATc1/Cx37
Figure Legend Snippet: Segregated Foxc2 and NFATc1 expression in developing peripheral VVs correlates closely with polarized Cx expression. Sections of P7-P11 VVs were triple immunostained for Foxc2/Cx37/Cx43 (A); NFATc1/Cx47/Cx37 (D); NFATc1/Cx43/Cx37 (E); Foxc2/NFATc1/Cx37

Techniques Used: Expressing

39) Product Images from "Segregated Foxc2, NFATc1 and Connexin expression at normal developing venous valves, and Connexin-specific differences in the valve phenotypes of Cx37, Cx43, and Cx47 knockout mice"

Article Title: Segregated Foxc2, NFATc1 and Connexin expression at normal developing venous valves, and Connexin-specific differences in the valve phenotypes of Cx37, Cx43, and Cx47 knockout mice

Journal: Developmental biology

doi: 10.1016/j.ydbio.2016.02.033

Segregated Foxc2 and NFATc1 expression in developing peripheral VVs correlates closely with polarized Cx expression. Sections of P7-P11 VVs were triple immunostained for Foxc2/Cx37/Cx43 (A); NFATc1/Cx47/Cx37 (D); NFATc1/Cx43/Cx37 (E); Foxc2/NFATc1/Cx37
Figure Legend Snippet: Segregated Foxc2 and NFATc1 expression in developing peripheral VVs correlates closely with polarized Cx expression. Sections of P7-P11 VVs were triple immunostained for Foxc2/Cx37/Cx43 (A); NFATc1/Cx47/Cx37 (D); NFATc1/Cx43/Cx37 (E); Foxc2/NFATc1/Cx37

Techniques Used: Expressing

40) Product Images from "Endocardial-to-mesenchymal transformation and mesenchymal cell colonization at the onset of human cardiac valve development"

Article Title: Endocardial-to-mesenchymal transformation and mesenchymal cell colonization at the onset of human cardiac valve development

Journal: Development (Cambridge, England)

doi: 10.1242/dev.133843

The spatiotemporal patterns of NFATC1 gene and protein expression. (A) Gene expression of NFATC1 in first and second trimester semilunar valve leaflets (* P
Figure Legend Snippet: The spatiotemporal patterns of NFATC1 gene and protein expression. (A) Gene expression of NFATC1 in first and second trimester semilunar valve leaflets (* P

Techniques Used: Expressing

Increased expression of VEGF targets in the first trimester. (A) Heat map representing the relative expression and fold enrichment of genes repressed by VEGF (VEGFA) in the first ( n =4) and second ( n =3) trimester in OFT leaflets (created using Molecular Signatures Database v5.0). See B for color key. (B) GSEA of enrichment of VEGF repression from leaflets obtained from the first compared with the second trimester. The false discovery rate (FDR) q-value and normalized enrichment score (NES) are shown. (C-L) Immunofluorescence imaging of NFATC1 (red) and VEGF (green) protein expression patterns in early endocardial cushions and elongated leaflets. DAPI is in white. At the early cushion stage (C,D), fibrosa and ventricularis are not yet identifiable. E-H, the fibrosa layer; I-L, the ventricularis. Scale bar: 50 µm.
Figure Legend Snippet: Increased expression of VEGF targets in the first trimester. (A) Heat map representing the relative expression and fold enrichment of genes repressed by VEGF (VEGFA) in the first ( n =4) and second ( n =3) trimester in OFT leaflets (created using Molecular Signatures Database v5.0). See B for color key. (B) GSEA of enrichment of VEGF repression from leaflets obtained from the first compared with the second trimester. The false discovery rate (FDR) q-value and normalized enrichment score (NES) are shown. (C-L) Immunofluorescence imaging of NFATC1 (red) and VEGF (green) protein expression patterns in early endocardial cushions and elongated leaflets. DAPI is in white. At the early cushion stage (C,D), fibrosa and ventricularis are not yet identifiable. E-H, the fibrosa layer; I-L, the ventricularis. Scale bar: 50 µm.

Techniques Used: Expressing, Immunofluorescence, Imaging

Related Articles

Activation Assay:

Article Title: Gene Expression Profiling of NFATc1-Knockdown in RAW 264.7 Cells: An Alternative Pathway for Macrophage Differentiation
Article Snippet: .. However, the observed activation of the transcription factors STAT6 and GATA2 in the absence of NFATc1 are worthy of further studies. ..

Article Title: Molecular Signaling Pathways Mediating Osteoclastogenesis Induced by Prostate Cancer Cells
Article Snippet: .. It has been well documented that RANKL stimulates calcium oscillations, resulting in sustained activation and up regulation of NFATc1 required for osteoclast differentiation [ , ]. .. In addition, we have previously shown that breast cancer cells produce factors capable of inducing calcium signaling and maintaining NFATc1 activation in RANKL-primed osteoclast precursors [ , , ].

Cell Differentiation:

Article Title: Gene Expression Profiling of NFATc1-Knockdown in RAW 264.7 Cells: An Alternative Pathway for Macrophage Differentiation
Article Snippet: .. In our bioinformatics analyses, GATA2 belonged to the GO group of “cell differentiation” and, most importantly, it was a central node in the protein network analysis by IPA , closely related to NFATc1 and STAT family members. .. GATA2 is considered an inducer for the reprogramming of cell pluripotency and belongs to a family of six transcription factors with well-established roles in cellular differentiation.

Indirect Immunoperoxidase Assay:

Article Title: Gene Expression Profiling of NFATc1-Knockdown in RAW 264.7 Cells: An Alternative Pathway for Macrophage Differentiation
Article Snippet: .. In our bioinformatics analyses, GATA2 belonged to the GO group of “cell differentiation” and, most importantly, it was a central node in the protein network analysis by IPA , closely related to NFATc1 and STAT family members. .. GATA2 is considered an inducer for the reprogramming of cell pluripotency and belongs to a family of six transcription factors with well-established roles in cellular differentiation.

Incubation:

Article Title: Effects of salubrinal on development of osteoclasts and osteoblasts from bone marrow-derived cells
Article Snippet: .. Downregulation of NFATc1 by salubrinal in bone marrow-derived cells and RAW264.7 pre-osteoclast cells Bone marrow-derived cells were incubated with RANKL in the presence and absence of salubrinal. .. Incubation with 20 ng/ml RANKL markedly increased the level of NFATc1, a master transcription factor for development of osteoclasts, and administration of 1 μM salubrinal reduced the RANKL-driven increase in NFATc1 by 24% (Figure A).

other:

Article Title: Molecular Signaling Pathways Mediating Osteoclastogenesis Induced by Prostate Cancer Cells
Article Snippet: Since NFATc1 is a calcium-dependent osteoclastogenic transcription factor, highly up-regulated during osteoclast formation [ , ], and involved in breast cancer-induced osteoclastogenesis [ ]; we next examined if NFATc1 mediates the osteoclastogenic effects of prostate cancer CM.

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

Article Title: Gene Expression Profiling of NFATc1-Knockdown in RAW 264.7 Cells: An Alternative Pathway for Macrophage Differentiation
Article Snippet: In fact, as we have already discussed, NFATc1 is ubiquitous and is involved in many differentiation programs other than osteoclastogenesis, whereas MITF-E is present only in the osteoclasts.

Expressing:

Article Title: Gene Expression Profiling of NFATc1-Knockdown in RAW 264.7 Cells: An Alternative Pathway for Macrophage Differentiation
Article Snippet: .. Our data strongly suggests that GATA2 is a downstream target of NFATc1, and we might speculate that NFATc1 possibly represses its expression through miRNA induction. ..

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    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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    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