tnf α  (Thermo Fisher)


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    Name:
    Tumor Necrosis Factor Alpha Recombinant Protein
    Description:
    Tumor Necrosis Factor Alpha Recombinant Protein for Western Blot ELISA
    Catalog Number:
    stnfa
    Price:
    None
    Applications:
    Protein Assays and Analysis|Protein Biology
    Category:
    Proteins Enzymes Peptides
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    Structured Review

    Thermo Fisher tnf α
    Effect of carnosine on gene expression. <t>TNF-α</t> stimulated HUVECS were exposed to 0 µM, 400 µM or 800 µM methylglyoxal (MGO) in the presence or absence of carnosine (CN, 20 mM) for 24 hours. Gene expression profiling was performed by microarray analysis. Displayed are the p -value distribution across all examined genes ( A , C ) and the corresponding volcano plot ( B and D ) for the comparisons 0 µM MGO no CN vs. 0 µM MGO + 20 mM CN (upper panel) and 800 µM MGO no CN vs. 800 µM MGO + 20 mM CN (lower panel). ( A ) The p -value distribution across all genes shows a homogenous pattern indicating that CN did not significantly affect the gene expression profile of TNF-α stimulated HUVECs. ( B ) The corresponding volcano plot analysis revealed only three genes to be significantly affected by CN as compared to control. ( C ) The p -value distribution shows a relative excess of p -values
    Tumor Necrosis Factor Alpha Recombinant Protein for Western Blot ELISA
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    tnf α - by Bioz Stars, 2020-11
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    Images

    1) Product Images from "Methylglyoxal down-regulates the expression of cell cycle associated genes and activates the p53 pathway in human umbilical vein endothelial cells"

    Article Title: Methylglyoxal down-regulates the expression of cell cycle associated genes and activates the p53 pathway in human umbilical vein endothelial cells

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-37937-1

    Effect of carnosine on gene expression. TNF-α stimulated HUVECS were exposed to 0 µM, 400 µM or 800 µM methylglyoxal (MGO) in the presence or absence of carnosine (CN, 20 mM) for 24 hours. Gene expression profiling was performed by microarray analysis. Displayed are the p -value distribution across all examined genes ( A , C ) and the corresponding volcano plot ( B and D ) for the comparisons 0 µM MGO no CN vs. 0 µM MGO + 20 mM CN (upper panel) and 800 µM MGO no CN vs. 800 µM MGO + 20 mM CN (lower panel). ( A ) The p -value distribution across all genes shows a homogenous pattern indicating that CN did not significantly affect the gene expression profile of TNF-α stimulated HUVECs. ( B ) The corresponding volcano plot analysis revealed only three genes to be significantly affected by CN as compared to control. ( C ) The p -value distribution shows a relative excess of p -values
    Figure Legend Snippet: Effect of carnosine on gene expression. TNF-α stimulated HUVECS were exposed to 0 µM, 400 µM or 800 µM methylglyoxal (MGO) in the presence or absence of carnosine (CN, 20 mM) for 24 hours. Gene expression profiling was performed by microarray analysis. Displayed are the p -value distribution across all examined genes ( A , C ) and the corresponding volcano plot ( B and D ) for the comparisons 0 µM MGO no CN vs. 0 µM MGO + 20 mM CN (upper panel) and 800 µM MGO no CN vs. 800 µM MGO + 20 mM CN (lower panel). ( A ) The p -value distribution across all genes shows a homogenous pattern indicating that CN did not significantly affect the gene expression profile of TNF-α stimulated HUVECs. ( B ) The corresponding volcano plot analysis revealed only three genes to be significantly affected by CN as compared to control. ( C ) The p -value distribution shows a relative excess of p -values

    Techniques Used: Expressing, Microarray

    Effect of 800 µM methylglyoxal on gene expression. TNF-α stimulated HUVECS were exposed to 0 µM, 400 µM or 800 µM methylglyoxal (MGO) in the presence or absence of carnosine (CN, 20 mM) for 24 hours. Gene expression profiling was performed by microarray analysis. Displayed are the p -value distribution across all examined genes ( A ) and the corresponding volcano plot ( B ) for the comparison 0 µM MGO vs. 800 µM MGO. ( A ) The distribution of p -values across all examined genes indicates an excess of p -values
    Figure Legend Snippet: Effect of 800 µM methylglyoxal on gene expression. TNF-α stimulated HUVECS were exposed to 0 µM, 400 µM or 800 µM methylglyoxal (MGO) in the presence or absence of carnosine (CN, 20 mM) for 24 hours. Gene expression profiling was performed by microarray analysis. Displayed are the p -value distribution across all examined genes ( A ) and the corresponding volcano plot ( B ) for the comparison 0 µM MGO vs. 800 µM MGO. ( A ) The distribution of p -values across all examined genes indicates an excess of p -values

    Techniques Used: Expressing, Microarray

    Differential gene expression by 800 µM methylglyoxal is compatible with cell cycle disruption and apoptosis. TNF-α stimulated HUVECS were exposed to 0 µM, 400 µM or 800 µM methylglyoxal (MGO) in the presence or absence of carnosine (CN, 20 mM) for 24 hours. Gene expression profiling was performed by microarray analysis. All differentially expressed genes (DEG, p adj
    Figure Legend Snippet: Differential gene expression by 800 µM methylglyoxal is compatible with cell cycle disruption and apoptosis. TNF-α stimulated HUVECS were exposed to 0 µM, 400 µM or 800 µM methylglyoxal (MGO) in the presence or absence of carnosine (CN, 20 mM) for 24 hours. Gene expression profiling was performed by microarray analysis. All differentially expressed genes (DEG, p adj

    Techniques Used: Expressing, Microarray

    Influence of carnosine on MGO-induced differential gene expression. TNF-α stimulated HUVECS were exposed to 0 µM, 400 µM or 800 µM methylglyoxal (MGO) in the presence or absence of carnosine (CN, 20 mM) for 24 hours. Gene expression profiling was performed by microarray analysis. All pie charts depict, how many of a selected number of genes are synergistically/antagonistically regulated genes by CN + 800 µM MGO vs. only 800 µM MGO (both in presence of TNF-α). ( A ) The upper Venn-diagram displays differentially expressed genes (DEG, p adj
    Figure Legend Snippet: Influence of carnosine on MGO-induced differential gene expression. TNF-α stimulated HUVECS were exposed to 0 µM, 400 µM or 800 µM methylglyoxal (MGO) in the presence or absence of carnosine (CN, 20 mM) for 24 hours. Gene expression profiling was performed by microarray analysis. All pie charts depict, how many of a selected number of genes are synergistically/antagonistically regulated genes by CN + 800 µM MGO vs. only 800 µM MGO (both in presence of TNF-α). ( A ) The upper Venn-diagram displays differentially expressed genes (DEG, p adj

    Techniques Used: Expressing, Microarray

    Methylglyoxal increased HO-1 expression, but diminished TNF-α induced VCAM-1 expression. HUVECs were stimulated with different combinations of methylglyoxal (MGO, 0 µM, 400 µM and 800 µM), TNF-α (12.5 ng/ml) and carnosine (CN, 20 mM). Western blotting and qPCR regarding HO-1 and VCAM-1 were performed after protein and RNA isolation. ( A ) HO-1 Western blotting demonstrates dose-dependent increase of HO-1 expression by MGO, which was abrogated by CN. ( B ) VCAM-1 Western blotting shows a clear TNF-α induced increase of the negligible baseline VCAM-1 signal, which was dose-dependently reduced by MGO. Equal protein loading was demonstrated by staining for β-actin. Displayed are the cropped blots. ( A , B ) show two independent experiments. The black surrounding lines demarcate individual blots. The scanned full-length blots are provided in Supplementary Figs 1 and 2 . ( C ) HO-1 densitometry (figure to the left) and gene expression (figure to the right) show a significant and dose dependent increase of HO-1 quantity, which was significantly reversed by CN. Protein and mRNA expression were normalized to β - actin. ( D ) VCAM-1 densitometry (figure to the left) and gene expression (figure to the right) demonstrate a significant, dose-dependent reduction of VCAM-1 by MGO at the protein, but not at the mRNA level. Carnosine did not significantly affect VCAM-1 quantity. Protein and mRNA expression were normalized to β - actin. For C and D data were analyzed using two-way ANOVA followed by Turkey’s multiple correction test. A p -value
    Figure Legend Snippet: Methylglyoxal increased HO-1 expression, but diminished TNF-α induced VCAM-1 expression. HUVECs were stimulated with different combinations of methylglyoxal (MGO, 0 µM, 400 µM and 800 µM), TNF-α (12.5 ng/ml) and carnosine (CN, 20 mM). Western blotting and qPCR regarding HO-1 and VCAM-1 were performed after protein and RNA isolation. ( A ) HO-1 Western blotting demonstrates dose-dependent increase of HO-1 expression by MGO, which was abrogated by CN. ( B ) VCAM-1 Western blotting shows a clear TNF-α induced increase of the negligible baseline VCAM-1 signal, which was dose-dependently reduced by MGO. Equal protein loading was demonstrated by staining for β-actin. Displayed are the cropped blots. ( A , B ) show two independent experiments. The black surrounding lines demarcate individual blots. The scanned full-length blots are provided in Supplementary Figs 1 and 2 . ( C ) HO-1 densitometry (figure to the left) and gene expression (figure to the right) show a significant and dose dependent increase of HO-1 quantity, which was significantly reversed by CN. Protein and mRNA expression were normalized to β - actin. ( D ) VCAM-1 densitometry (figure to the left) and gene expression (figure to the right) demonstrate a significant, dose-dependent reduction of VCAM-1 by MGO at the protein, but not at the mRNA level. Carnosine did not significantly affect VCAM-1 quantity. Protein and mRNA expression were normalized to β - actin. For C and D data were analyzed using two-way ANOVA followed by Turkey’s multiple correction test. A p -value

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

    2) Product Images from "Methylglyoxal down-regulates the expression of cell cycle associated genes and activates the p53 pathway in human umbilical vein endothelial cells"

    Article Title: Methylglyoxal down-regulates the expression of cell cycle associated genes and activates the p53 pathway in human umbilical vein endothelial cells

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-37937-1

    Effect of carnosine on gene expression. TNF-α stimulated HUVECS were exposed to 0 µM, 400 µM or 800 µM methylglyoxal (MGO) in the presence or absence of carnosine (CN, 20 mM) for 24 hours. Gene expression profiling was performed by microarray analysis. Displayed are the p -value distribution across all examined genes ( A , C ) and the corresponding volcano plot ( B and D ) for the comparisons 0 µM MGO no CN vs. 0 µM MGO + 20 mM CN (upper panel) and 800 µM MGO no CN vs. 800 µM MGO + 20 mM CN (lower panel). ( A ) The p -value distribution across all genes shows a homogenous pattern indicating that CN did not significantly affect the gene expression profile of TNF-α stimulated HUVECs. ( B ) The corresponding volcano plot analysis revealed only three genes to be significantly affected by CN as compared to control. ( C ) The p -value distribution shows a relative excess of p -values
    Figure Legend Snippet: Effect of carnosine on gene expression. TNF-α stimulated HUVECS were exposed to 0 µM, 400 µM or 800 µM methylglyoxal (MGO) in the presence or absence of carnosine (CN, 20 mM) for 24 hours. Gene expression profiling was performed by microarray analysis. Displayed are the p -value distribution across all examined genes ( A , C ) and the corresponding volcano plot ( B and D ) for the comparisons 0 µM MGO no CN vs. 0 µM MGO + 20 mM CN (upper panel) and 800 µM MGO no CN vs. 800 µM MGO + 20 mM CN (lower panel). ( A ) The p -value distribution across all genes shows a homogenous pattern indicating that CN did not significantly affect the gene expression profile of TNF-α stimulated HUVECs. ( B ) The corresponding volcano plot analysis revealed only three genes to be significantly affected by CN as compared to control. ( C ) The p -value distribution shows a relative excess of p -values

    Techniques Used: Expressing, Microarray

    Effect of 800 µM methylglyoxal on gene expression. TNF-α stimulated HUVECS were exposed to 0 µM, 400 µM or 800 µM methylglyoxal (MGO) in the presence or absence of carnosine (CN, 20 mM) for 24 hours. Gene expression profiling was performed by microarray analysis. Displayed are the p -value distribution across all examined genes ( A ) and the corresponding volcano plot ( B ) for the comparison 0 µM MGO vs. 800 µM MGO. ( A ) The distribution of p -values across all examined genes indicates an excess of p -values
    Figure Legend Snippet: Effect of 800 µM methylglyoxal on gene expression. TNF-α stimulated HUVECS were exposed to 0 µM, 400 µM or 800 µM methylglyoxal (MGO) in the presence or absence of carnosine (CN, 20 mM) for 24 hours. Gene expression profiling was performed by microarray analysis. Displayed are the p -value distribution across all examined genes ( A ) and the corresponding volcano plot ( B ) for the comparison 0 µM MGO vs. 800 µM MGO. ( A ) The distribution of p -values across all examined genes indicates an excess of p -values

    Techniques Used: Expressing, Microarray

    Differential gene expression by 800 µM methylglyoxal is compatible with cell cycle disruption and apoptosis. TNF-α stimulated HUVECS were exposed to 0 µM, 400 µM or 800 µM methylglyoxal (MGO) in the presence or absence of carnosine (CN, 20 mM) for 24 hours. Gene expression profiling was performed by microarray analysis. All differentially expressed genes (DEG, p adj
    Figure Legend Snippet: Differential gene expression by 800 µM methylglyoxal is compatible with cell cycle disruption and apoptosis. TNF-α stimulated HUVECS were exposed to 0 µM, 400 µM or 800 µM methylglyoxal (MGO) in the presence or absence of carnosine (CN, 20 mM) for 24 hours. Gene expression profiling was performed by microarray analysis. All differentially expressed genes (DEG, p adj

    Techniques Used: Expressing, Microarray

    Influence of carnosine on MGO-induced differential gene expression. TNF-α stimulated HUVECS were exposed to 0 µM, 400 µM or 800 µM methylglyoxal (MGO) in the presence or absence of carnosine (CN, 20 mM) for 24 hours. Gene expression profiling was performed by microarray analysis. All pie charts depict, how many of a selected number of genes are synergistically/antagonistically regulated genes by CN + 800 µM MGO vs. only 800 µM MGO (both in presence of TNF-α). ( A ) The upper Venn-diagram displays differentially expressed genes (DEG, p adj
    Figure Legend Snippet: Influence of carnosine on MGO-induced differential gene expression. TNF-α stimulated HUVECS were exposed to 0 µM, 400 µM or 800 µM methylglyoxal (MGO) in the presence or absence of carnosine (CN, 20 mM) for 24 hours. Gene expression profiling was performed by microarray analysis. All pie charts depict, how many of a selected number of genes are synergistically/antagonistically regulated genes by CN + 800 µM MGO vs. only 800 µM MGO (both in presence of TNF-α). ( A ) The upper Venn-diagram displays differentially expressed genes (DEG, p adj

    Techniques Used: Expressing, Microarray

    Methylglyoxal increased HO-1 expression, but diminished TNF-α induced VCAM-1 expression. HUVECs were stimulated with different combinations of methylglyoxal (MGO, 0 µM, 400 µM and 800 µM), TNF-α (12.5 ng/ml) and carnosine (CN, 20 mM). Western blotting and qPCR regarding HO-1 and VCAM-1 were performed after protein and RNA isolation. ( A ) HO-1 Western blotting demonstrates dose-dependent increase of HO-1 expression by MGO, which was abrogated by CN. ( B ) VCAM-1 Western blotting shows a clear TNF-α induced increase of the negligible baseline VCAM-1 signal, which was dose-dependently reduced by MGO. Equal protein loading was demonstrated by staining for β-actin. Displayed are the cropped blots. ( A , B ) show two independent experiments. The black surrounding lines demarcate individual blots. The scanned full-length blots are provided in Supplementary Figs 1 and 2 . ( C ) HO-1 densitometry (figure to the left) and gene expression (figure to the right) show a significant and dose dependent increase of HO-1 quantity, which was significantly reversed by CN. Protein and mRNA expression were normalized to β - actin. ( D ) VCAM-1 densitometry (figure to the left) and gene expression (figure to the right) demonstrate a significant, dose-dependent reduction of VCAM-1 by MGO at the protein, but not at the mRNA level. Carnosine did not significantly affect VCAM-1 quantity. Protein and mRNA expression were normalized to β - actin. For C and D data were analyzed using two-way ANOVA followed by Turkey’s multiple correction test. A p -value
    Figure Legend Snippet: Methylglyoxal increased HO-1 expression, but diminished TNF-α induced VCAM-1 expression. HUVECs were stimulated with different combinations of methylglyoxal (MGO, 0 µM, 400 µM and 800 µM), TNF-α (12.5 ng/ml) and carnosine (CN, 20 mM). Western blotting and qPCR regarding HO-1 and VCAM-1 were performed after protein and RNA isolation. ( A ) HO-1 Western blotting demonstrates dose-dependent increase of HO-1 expression by MGO, which was abrogated by CN. ( B ) VCAM-1 Western blotting shows a clear TNF-α induced increase of the negligible baseline VCAM-1 signal, which was dose-dependently reduced by MGO. Equal protein loading was demonstrated by staining for β-actin. Displayed are the cropped blots. ( A , B ) show two independent experiments. The black surrounding lines demarcate individual blots. The scanned full-length blots are provided in Supplementary Figs 1 and 2 . ( C ) HO-1 densitometry (figure to the left) and gene expression (figure to the right) show a significant and dose dependent increase of HO-1 quantity, which was significantly reversed by CN. Protein and mRNA expression were normalized to β - actin. ( D ) VCAM-1 densitometry (figure to the left) and gene expression (figure to the right) demonstrate a significant, dose-dependent reduction of VCAM-1 by MGO at the protein, but not at the mRNA level. Carnosine did not significantly affect VCAM-1 quantity. Protein and mRNA expression were normalized to β - actin. For C and D data were analyzed using two-way ANOVA followed by Turkey’s multiple correction test. A p -value

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

    3) Product Images from "Methylglyoxal down-regulates the expression of cell cycle associated genes and activates the p53 pathway in human umbilical vein endothelial cells"

    Article Title: Methylglyoxal down-regulates the expression of cell cycle associated genes and activates the p53 pathway in human umbilical vein endothelial cells

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-37937-1

    Effect of carnosine on gene expression. TNF-α stimulated HUVECS were exposed to 0 µM, 400 µM or 800 µM methylglyoxal (MGO) in the presence or absence of carnosine (CN, 20 mM) for 24 hours. Gene expression profiling was performed by microarray analysis. Displayed are the p -value distribution across all examined genes ( A , C ) and the corresponding volcano plot ( B and D ) for the comparisons 0 µM MGO no CN vs. 0 µM MGO + 20 mM CN (upper panel) and 800 µM MGO no CN vs. 800 µM MGO + 20 mM CN (lower panel). ( A ) The p -value distribution across all genes shows a homogenous pattern indicating that CN did not significantly affect the gene expression profile of TNF-α stimulated HUVECs. ( B ) The corresponding volcano plot analysis revealed only three genes to be significantly affected by CN as compared to control. ( C ) The p -value distribution shows a relative excess of p -values
    Figure Legend Snippet: Effect of carnosine on gene expression. TNF-α stimulated HUVECS were exposed to 0 µM, 400 µM or 800 µM methylglyoxal (MGO) in the presence or absence of carnosine (CN, 20 mM) for 24 hours. Gene expression profiling was performed by microarray analysis. Displayed are the p -value distribution across all examined genes ( A , C ) and the corresponding volcano plot ( B and D ) for the comparisons 0 µM MGO no CN vs. 0 µM MGO + 20 mM CN (upper panel) and 800 µM MGO no CN vs. 800 µM MGO + 20 mM CN (lower panel). ( A ) The p -value distribution across all genes shows a homogenous pattern indicating that CN did not significantly affect the gene expression profile of TNF-α stimulated HUVECs. ( B ) The corresponding volcano plot analysis revealed only three genes to be significantly affected by CN as compared to control. ( C ) The p -value distribution shows a relative excess of p -values

    Techniques Used: Expressing, Microarray

    Effect of 800 µM methylglyoxal on gene expression. TNF-α stimulated HUVECS were exposed to 0 µM, 400 µM or 800 µM methylglyoxal (MGO) in the presence or absence of carnosine (CN, 20 mM) for 24 hours. Gene expression profiling was performed by microarray analysis. Displayed are the p -value distribution across all examined genes ( A ) and the corresponding volcano plot ( B ) for the comparison 0 µM MGO vs. 800 µM MGO. ( A ) The distribution of p -values across all examined genes indicates an excess of p -values
    Figure Legend Snippet: Effect of 800 µM methylglyoxal on gene expression. TNF-α stimulated HUVECS were exposed to 0 µM, 400 µM or 800 µM methylglyoxal (MGO) in the presence or absence of carnosine (CN, 20 mM) for 24 hours. Gene expression profiling was performed by microarray analysis. Displayed are the p -value distribution across all examined genes ( A ) and the corresponding volcano plot ( B ) for the comparison 0 µM MGO vs. 800 µM MGO. ( A ) The distribution of p -values across all examined genes indicates an excess of p -values

    Techniques Used: Expressing, Microarray

    Differential gene expression by 800 µM methylglyoxal is compatible with cell cycle disruption and apoptosis. TNF-α stimulated HUVECS were exposed to 0 µM, 400 µM or 800 µM methylglyoxal (MGO) in the presence or absence of carnosine (CN, 20 mM) for 24 hours. Gene expression profiling was performed by microarray analysis. All differentially expressed genes (DEG, p adj
    Figure Legend Snippet: Differential gene expression by 800 µM methylglyoxal is compatible with cell cycle disruption and apoptosis. TNF-α stimulated HUVECS were exposed to 0 µM, 400 µM or 800 µM methylglyoxal (MGO) in the presence or absence of carnosine (CN, 20 mM) for 24 hours. Gene expression profiling was performed by microarray analysis. All differentially expressed genes (DEG, p adj

    Techniques Used: Expressing, Microarray

    Influence of carnosine on MGO-induced differential gene expression. TNF-α stimulated HUVECS were exposed to 0 µM, 400 µM or 800 µM methylglyoxal (MGO) in the presence or absence of carnosine (CN, 20 mM) for 24 hours. Gene expression profiling was performed by microarray analysis. All pie charts depict, how many of a selected number of genes are synergistically/antagonistically regulated genes by CN + 800 µM MGO vs. only 800 µM MGO (both in presence of TNF-α). ( A ) The upper Venn-diagram displays differentially expressed genes (DEG, p adj
    Figure Legend Snippet: Influence of carnosine on MGO-induced differential gene expression. TNF-α stimulated HUVECS were exposed to 0 µM, 400 µM or 800 µM methylglyoxal (MGO) in the presence or absence of carnosine (CN, 20 mM) for 24 hours. Gene expression profiling was performed by microarray analysis. All pie charts depict, how many of a selected number of genes are synergistically/antagonistically regulated genes by CN + 800 µM MGO vs. only 800 µM MGO (both in presence of TNF-α). ( A ) The upper Venn-diagram displays differentially expressed genes (DEG, p adj

    Techniques Used: Expressing, Microarray

    Methylglyoxal increased HO-1 expression, but diminished TNF-α induced VCAM-1 expression. HUVECs were stimulated with different combinations of methylglyoxal (MGO, 0 µM, 400 µM and 800 µM), TNF-α (12.5 ng/ml) and carnosine (CN, 20 mM). Western blotting and qPCR regarding HO-1 and VCAM-1 were performed after protein and RNA isolation. ( A ) HO-1 Western blotting demonstrates dose-dependent increase of HO-1 expression by MGO, which was abrogated by CN. ( B ) VCAM-1 Western blotting shows a clear TNF-α induced increase of the negligible baseline VCAM-1 signal, which was dose-dependently reduced by MGO. Equal protein loading was demonstrated by staining for β-actin. Displayed are the cropped blots. ( A , B ) show two independent experiments. The black surrounding lines demarcate individual blots. The scanned full-length blots are provided in Supplementary Figs 1 and 2 . ( C ) HO-1 densitometry (figure to the left) and gene expression (figure to the right) show a significant and dose dependent increase of HO-1 quantity, which was significantly reversed by CN. Protein and mRNA expression were normalized to β - actin. ( D ) VCAM-1 densitometry (figure to the left) and gene expression (figure to the right) demonstrate a significant, dose-dependent reduction of VCAM-1 by MGO at the protein, but not at the mRNA level. Carnosine did not significantly affect VCAM-1 quantity. Protein and mRNA expression were normalized to β - actin. For C and D data were analyzed using two-way ANOVA followed by Turkey’s multiple correction test. A p -value
    Figure Legend Snippet: Methylglyoxal increased HO-1 expression, but diminished TNF-α induced VCAM-1 expression. HUVECs were stimulated with different combinations of methylglyoxal (MGO, 0 µM, 400 µM and 800 µM), TNF-α (12.5 ng/ml) and carnosine (CN, 20 mM). Western blotting and qPCR regarding HO-1 and VCAM-1 were performed after protein and RNA isolation. ( A ) HO-1 Western blotting demonstrates dose-dependent increase of HO-1 expression by MGO, which was abrogated by CN. ( B ) VCAM-1 Western blotting shows a clear TNF-α induced increase of the negligible baseline VCAM-1 signal, which was dose-dependently reduced by MGO. Equal protein loading was demonstrated by staining for β-actin. Displayed are the cropped blots. ( A , B ) show two independent experiments. The black surrounding lines demarcate individual blots. The scanned full-length blots are provided in Supplementary Figs 1 and 2 . ( C ) HO-1 densitometry (figure to the left) and gene expression (figure to the right) show a significant and dose dependent increase of HO-1 quantity, which was significantly reversed by CN. Protein and mRNA expression were normalized to β - actin. ( D ) VCAM-1 densitometry (figure to the left) and gene expression (figure to the right) demonstrate a significant, dose-dependent reduction of VCAM-1 by MGO at the protein, but not at the mRNA level. Carnosine did not significantly affect VCAM-1 quantity. Protein and mRNA expression were normalized to β - actin. For C and D data were analyzed using two-way ANOVA followed by Turkey’s multiple correction test. A p -value

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

    4) Product Images from "Herpes Simplex Virus 1 UL2 Inhibits the TNF-α–Mediated NF-κB Activity by Interacting With p65/p50"

    Article Title: Herpes Simplex Virus 1 UL2 Inhibits the TNF-α–Mediated NF-κB Activity by Interacting With p65/p50

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2020.00549

    HSV-1 UL2 does not block the TNF-α–induced nuclear translocation of p65 or p50. HeLa cells were transfected with HA vector or UL2-HA expression plasmid. Twenty-four hours posttransfection, cells were treated with TNF-α (10 ng/mL) or mock-treated for 30 min. Then, cells were stained with anti-HA mAb and anti-p65 pAb (A) or anti-p50 pAb (B) . Fluorescein isothiocyanate–conjugated donkey anti–mouse IgG (green) and Cy5-conjugated goat anti–rabbit IgG (red) were used as the secondary Abs. Cell nuclei were stained with DAPI (blue). All of the transfected cells were analyzed by a confocal microscope (Axio-Imager-LSM-800; Zeiss), and the photomicrographs were taken at a magnification of 400×. Each image represented a vast majority of the cells with similar subcellular distribution. Statistical analysis of the subcellular localization of p65 or p50 in the absence or presence of UL2 is shown in Table 2 .
    Figure Legend Snippet: HSV-1 UL2 does not block the TNF-α–induced nuclear translocation of p65 or p50. HeLa cells were transfected with HA vector or UL2-HA expression plasmid. Twenty-four hours posttransfection, cells were treated with TNF-α (10 ng/mL) or mock-treated for 30 min. Then, cells were stained with anti-HA mAb and anti-p65 pAb (A) or anti-p50 pAb (B) . Fluorescein isothiocyanate–conjugated donkey anti–mouse IgG (green) and Cy5-conjugated goat anti–rabbit IgG (red) were used as the secondary Abs. Cell nuclei were stained with DAPI (blue). All of the transfected cells were analyzed by a confocal microscope (Axio-Imager-LSM-800; Zeiss), and the photomicrographs were taken at a magnification of 400×. Each image represented a vast majority of the cells with similar subcellular distribution. Statistical analysis of the subcellular localization of p65 or p50 in the absence or presence of UL2 is shown in Table 2 .

    Techniques Used: Blocking Assay, Translocation Assay, Transfection, Plasmid Preparation, Expressing, Staining, Microscopy

    p65 phosphorylation at Ser536 is suppressed by HSV-1 UL2. (A,C) HEK293T cells transfected with either HA empty vector or UL2-HA expression plasmid were stimulated with TNF-α (20 ng/mL) for the indicated times (0, 30, and 60 min) according to previous studies ( 59 , 77 ), and then equal amounts of cell lysates were analyzed by WBs with phospho-NF-κB–p65 (Ser536) Ab (A) , phospho-NF-κB–p65 (Ser276) Ab (C) (top panel), or anti-p65 pAb (second panel). Protein levels of UL2 (third panel) and β-actin (bottom panel) in the same cell lysates were also determined. (B,D) Densitometry of phospho-NF-κB–p65 Ser536 (B) and Ser276 bands (D) from (A,C) , respectively, were normalized to loading control β-actin. Data were expressed as means ± SD from three independent experiments. ns, not significant and *** P
    Figure Legend Snippet: p65 phosphorylation at Ser536 is suppressed by HSV-1 UL2. (A,C) HEK293T cells transfected with either HA empty vector or UL2-HA expression plasmid were stimulated with TNF-α (20 ng/mL) for the indicated times (0, 30, and 60 min) according to previous studies ( 59 , 77 ), and then equal amounts of cell lysates were analyzed by WBs with phospho-NF-κB–p65 (Ser536) Ab (A) , phospho-NF-κB–p65 (Ser276) Ab (C) (top panel), or anti-p65 pAb (second panel). Protein levels of UL2 (third panel) and β-actin (bottom panel) in the same cell lysates were also determined. (B,D) Densitometry of phospho-NF-κB–p65 Ser536 (B) and Ser276 bands (D) from (A,C) , respectively, were normalized to loading control β-actin. Data were expressed as means ± SD from three independent experiments. ns, not significant and *** P

    Techniques Used: Transfection, Plasmid Preparation, Expressing

    Herpes simplex virus 1 UL2 inhibits NF-κB–driven cytokine expression. (A) HEK293T cells were cotransfected with Flag vector or diverse concentrations (100, 250, and 500 ng) of UL2-Flag expression plasmid along with reporter plasmids pXP2-pIL-8-Luc and pRL-TK. Twenty-four hours posttransfection, cells were treated with TNF-α (10 ng/mL) for 6 h, and luciferase activity was measured as described in Figure 1 . (B) HEK293T cells were transfected with 1 μg of HA control vector or UL2-HA expression plasmid; 24 h posttransfection, cells were treated with TNF-α (10 ng/mL) for 6 h, and then RT-qPCR analysis was performed to analyze the relative expression level of IL-8 mRNA. Glyceraldehyde-3-phosphate dehydrogenase was used as the housekeeping gene. (C) was carried out as (B) , except that HeLa cells were used for transfection. The expression of UL2 was analyzed by WB using anti-Flag mAb or anti-HA mAb, and β-actin was used to verify equal loading of protein in each lane. (D) HEK293T cells were mock-infected or infected with WT, UL2 Del, or UL2 Rev HSV-1 BAC GFP Luc virus at an MOI of 1 for 16 h. Flow cytometry analysis was then carried out to detect the GFP fluorescence. (E) HEK293T cells were infected with WT, UL2 Del, or UL2 Rev HSV-1 BAC GFP Luc virus at an MOI of 1. Sixteen hours postinfection, cells were treated with TNF-α (10 ng/mL) for 6 h. Then, RT-qPCR analysis was performed to detect the relative expression level of IL-8 mRNA. Data were expressed as means ± SD from three independent experiments. * P
    Figure Legend Snippet: Herpes simplex virus 1 UL2 inhibits NF-κB–driven cytokine expression. (A) HEK293T cells were cotransfected with Flag vector or diverse concentrations (100, 250, and 500 ng) of UL2-Flag expression plasmid along with reporter plasmids pXP2-pIL-8-Luc and pRL-TK. Twenty-four hours posttransfection, cells were treated with TNF-α (10 ng/mL) for 6 h, and luciferase activity was measured as described in Figure 1 . (B) HEK293T cells were transfected with 1 μg of HA control vector or UL2-HA expression plasmid; 24 h posttransfection, cells were treated with TNF-α (10 ng/mL) for 6 h, and then RT-qPCR analysis was performed to analyze the relative expression level of IL-8 mRNA. Glyceraldehyde-3-phosphate dehydrogenase was used as the housekeeping gene. (C) was carried out as (B) , except that HeLa cells were used for transfection. The expression of UL2 was analyzed by WB using anti-Flag mAb or anti-HA mAb, and β-actin was used to verify equal loading of protein in each lane. (D) HEK293T cells were mock-infected or infected with WT, UL2 Del, or UL2 Rev HSV-1 BAC GFP Luc virus at an MOI of 1 for 16 h. Flow cytometry analysis was then carried out to detect the GFP fluorescence. (E) HEK293T cells were infected with WT, UL2 Del, or UL2 Rev HSV-1 BAC GFP Luc virus at an MOI of 1. Sixteen hours postinfection, cells were treated with TNF-α (10 ng/mL) for 6 h. Then, RT-qPCR analysis was performed to detect the relative expression level of IL-8 mRNA. Data were expressed as means ± SD from three independent experiments. * P

    Techniques Used: Expressing, Plasmid Preparation, Luciferase, Activity Assay, Transfection, Quantitative RT-PCR, Western Blot, Infection, BAC Assay, Flow Cytometry, Fluorescence

    The region of aa9-17 is responsible for HSV-1 UL2 inhibition of NF-κB activity through interacting with p65 and p50. (A) Schematic representations of WT and truncated mutants of UL2 constructed in our laboratory. (B,C) HEK293T cells were cotransfected with NF-κB–Luc and pRL-TK reporter plasmids, along with 500 ng of EYFP empty vector or plasmid encoding EYFP-fused WT or truncated mutants of UL2. Twenty-four hours posttransfection, cells were treated with or without TNF-α (10 ng/mL) for 6 h, and luciferase activity was analyzed, as described in Figure 1 . Data were expressed as means ± SD from three independent experiments. (D–N) HEK293T cells were cotransfected with pUL2(9-17)-EYFP and Flag vector (D) , pUL2(9-17)-EYFP and pCMV-p65-Flag (E) , pUL2(9-17)-EYFP and p50-Flag (F) , pUL2(278-334)-EYFP and pCMV-p65-Flag (G) , pUL2(278-334)-EYFP and p50-Flag (H) , pUL2(9-17)del-EYFP and pCMV-p65-Flag (I) , pUL2(9-17)del-EYFP and p50-Flag (J) , pUL2(69-75)-EYFP and pCMV-p65-Flag (K) , pUL2(69-75)-EYFP and p50-Flag (L) , pUL2(9-17)(69-75)del-EYFP and pCMV-p65-Flag (M) , or pUL2(9-17)(69-75)del-EYFP and p50-Flag (N) expression plasmids. Twenty-four hours posttransfection, cells were harvested and lysed, and the samples were then subjected to Co-IP assays using anti-Flag mAb, anti-EYFP or non-specific mouse IgG. Western blots were probed with the indicated Abs. ** P
    Figure Legend Snippet: The region of aa9-17 is responsible for HSV-1 UL2 inhibition of NF-κB activity through interacting with p65 and p50. (A) Schematic representations of WT and truncated mutants of UL2 constructed in our laboratory. (B,C) HEK293T cells were cotransfected with NF-κB–Luc and pRL-TK reporter plasmids, along with 500 ng of EYFP empty vector or plasmid encoding EYFP-fused WT or truncated mutants of UL2. Twenty-four hours posttransfection, cells were treated with or without TNF-α (10 ng/mL) for 6 h, and luciferase activity was analyzed, as described in Figure 1 . Data were expressed as means ± SD from three independent experiments. (D–N) HEK293T cells were cotransfected with pUL2(9-17)-EYFP and Flag vector (D) , pUL2(9-17)-EYFP and pCMV-p65-Flag (E) , pUL2(9-17)-EYFP and p50-Flag (F) , pUL2(278-334)-EYFP and pCMV-p65-Flag (G) , pUL2(278-334)-EYFP and p50-Flag (H) , pUL2(9-17)del-EYFP and pCMV-p65-Flag (I) , pUL2(9-17)del-EYFP and p50-Flag (J) , pUL2(69-75)-EYFP and pCMV-p65-Flag (K) , pUL2(69-75)-EYFP and p50-Flag (L) , pUL2(9-17)(69-75)del-EYFP and pCMV-p65-Flag (M) , or pUL2(9-17)(69-75)del-EYFP and p50-Flag (N) expression plasmids. Twenty-four hours posttransfection, cells were harvested and lysed, and the samples were then subjected to Co-IP assays using anti-Flag mAb, anti-EYFP or non-specific mouse IgG. Western blots were probed with the indicated Abs. ** P

    Techniques Used: Inhibition, Activity Assay, Construct, Plasmid Preparation, Luciferase, Expressing, Co-Immunoprecipitation Assay, Western Blot

    Herpes simplex virus 1 UL2 interacts with endogenous p65 and p50. (A,C) HEK293T cells were cotransfected with pCMV-p65-Flag and pUL2-HA (A) or p50-Flag and pUL2-HA (C) expression plasmids. Twenty-four hours posttransfection, cells were harvested and lysed, and the samples were then subjected to Co-IP assays using anti-Flag mAb or non-specific mouse IgG. Western blots were probed with the indicated Abs. (B,D) HEK293T cells were cotransfected with plasmids as described for (A,C) , respectively. Protein lysates were Co-IPed using anti-HA mAb, and WBs were analyzed with the indicated Abs. (E,F) HEK293T cells infected with WT HSV-1 at an MOI of 1 for 24 h were treated with 10 ng/mL of the TNF-α for an additional 6 h. Cells were then lysed, and the extracts were subjected to Co-IP using anti-p65 pAb (E) , anti-p50 pAb (F) , or control IgG. Samples were analyzed by WBs with the indicated Abs.
    Figure Legend Snippet: Herpes simplex virus 1 UL2 interacts with endogenous p65 and p50. (A,C) HEK293T cells were cotransfected with pCMV-p65-Flag and pUL2-HA (A) or p50-Flag and pUL2-HA (C) expression plasmids. Twenty-four hours posttransfection, cells were harvested and lysed, and the samples were then subjected to Co-IP assays using anti-Flag mAb or non-specific mouse IgG. Western blots were probed with the indicated Abs. (B,D) HEK293T cells were cotransfected with plasmids as described for (A,C) , respectively. Protein lysates were Co-IPed using anti-HA mAb, and WBs were analyzed with the indicated Abs. (E,F) HEK293T cells infected with WT HSV-1 at an MOI of 1 for 24 h were treated with 10 ng/mL of the TNF-α for an additional 6 h. Cells were then lysed, and the extracts were subjected to Co-IP using anti-p65 pAb (E) , anti-p50 pAb (F) , or control IgG. Samples were analyzed by WBs with the indicated Abs.

    Techniques Used: Expressing, Co-Immunoprecipitation Assay, Western Blot, Infection

    Inhibition of TNF-α–induced NF-κB activation by HSV-1 UL2. (A) HEK293T cells were transfected with promoter reporter plasmids NF-κB–Luc and pRL-TK, together with 500 ng of Flag empty vector or pUL2-Flag plasmid. Twenty-four hours posttransfection, cells were treated with or without 10 ng/mL of the recombinant human TNF-α and incubated for an additional 6 h, followed by cell lysed. Nuclear factor κB–driven luciferase activity was detected by DLR, as described in section “Materials and Methods.” (B) was carried out as (A) ; except that for an increase indicated amounts (100, 250, and 500 ng) of UL2-Flag expression plasmid were used. Cell lysates were divided into two aliquots; one aliquot was used for DLR detection, and the other was used for WB analysis to detect the protein expression of transfected plasmid. The expression of UL2 was analyzed by WB with anti-Flag mAb, and β-actin was used to verify equal loading of protein in each lane. Dual-luciferase reporter data were normalized for transfection efficiency through measuring firefly luciferase activity and Renilla luciferase activity, and values were shown as the ratio between the firefly and Renilla luciferase. Data were expressed as means ± SD from three independent experiments. *** P
    Figure Legend Snippet: Inhibition of TNF-α–induced NF-κB activation by HSV-1 UL2. (A) HEK293T cells were transfected with promoter reporter plasmids NF-κB–Luc and pRL-TK, together with 500 ng of Flag empty vector or pUL2-Flag plasmid. Twenty-four hours posttransfection, cells were treated with or without 10 ng/mL of the recombinant human TNF-α and incubated for an additional 6 h, followed by cell lysed. Nuclear factor κB–driven luciferase activity was detected by DLR, as described in section “Materials and Methods.” (B) was carried out as (A) ; except that for an increase indicated amounts (100, 250, and 500 ng) of UL2-Flag expression plasmid were used. Cell lysates were divided into two aliquots; one aliquot was used for DLR detection, and the other was used for WB analysis to detect the protein expression of transfected plasmid. The expression of UL2 was analyzed by WB with anti-Flag mAb, and β-actin was used to verify equal loading of protein in each lane. Dual-luciferase reporter data were normalized for transfection efficiency through measuring firefly luciferase activity and Renilla luciferase activity, and values were shown as the ratio between the firefly and Renilla luciferase. Data were expressed as means ± SD from three independent experiments. *** P

    Techniques Used: Inhibition, Activation Assay, Transfection, Plasmid Preparation, Recombinant, Incubation, Luciferase, Activity Assay, Expressing, Western Blot

    5) Product Images from "LCZ696 mitigates diabetic-induced nephropathy through inhibiting oxidative stress, NF-κB mediated inflammation and glomerulosclerosis in rats"

    Article Title: LCZ696 mitigates diabetic-induced nephropathy through inhibiting oxidative stress, NF-κB mediated inflammation and glomerulosclerosis in rats

    Journal: PeerJ

    doi: 10.7717/peerj.9196

    Effect of valsartan and LCZ696 on renal inflammatory bio-markers including tumor necrosis factor-α (TNF-α) (A), interleukin-1β (IL-1β) (B), interleukin-6 (IL-6) (C) and nuclear factor kappa-B (NF-κB) (D) in STZ-induced diabetic rats ( n = 6 per group). Statistically significant difference: ** p
    Figure Legend Snippet: Effect of valsartan and LCZ696 on renal inflammatory bio-markers including tumor necrosis factor-α (TNF-α) (A), interleukin-1β (IL-1β) (B), interleukin-6 (IL-6) (C) and nuclear factor kappa-B (NF-κB) (D) in STZ-induced diabetic rats ( n = 6 per group). Statistically significant difference: ** p

    Techniques Used:

    Effect of valsartan and LCZ696 on serum pro-inflammatory cytokines including interleukin-6 (IL-6) (A), interleukin-1β (IL-1β) (B), tumor necrosis factor-α (TNF-α) (C) and interleukin-10 (IL-10) (D) in STZ-induced diabetic rats. Data are expressed as the mean ± SEM ( n = 6 per group). Statistically significant difference: ** p
    Figure Legend Snippet: Effect of valsartan and LCZ696 on serum pro-inflammatory cytokines including interleukin-6 (IL-6) (A), interleukin-1β (IL-1β) (B), tumor necrosis factor-α (TNF-α) (C) and interleukin-10 (IL-10) (D) in STZ-induced diabetic rats. Data are expressed as the mean ± SEM ( n = 6 per group). Statistically significant difference: ** p

    Techniques Used:

    6) Product Images from "Mesenchymal Stem Cells Provide Neuroprotection by Regulating Heat Stroke-Induced Brain Inflammation"

    Article Title: Mesenchymal Stem Cells Provide Neuroprotection by Regulating Heat Stroke-Induced Brain Inflammation

    Journal: Frontiers in Neurology

    doi: 10.3389/fneur.2020.00372

    MSCs modulated pro-inflammatory and anti-inflammatory reactions of HS rats. At different time points after MSCs or saline infusion, the blood of rats in each group was collected, and the IL-10 (A) , IL-1β (B) , IL-6 (C) , and TNF-α (D) levels in the rat's blood serum were assayed. n = 10 rats per group. ** P
    Figure Legend Snippet: MSCs modulated pro-inflammatory and anti-inflammatory reactions of HS rats. At different time points after MSCs or saline infusion, the blood of rats in each group was collected, and the IL-10 (A) , IL-1β (B) , IL-6 (C) , and TNF-α (D) levels in the rat's blood serum were assayed. n = 10 rats per group. ** P

    Techniques Used:

    MSC administration modulates inflammatory and chemotactic cytokines in the brain tissue of HS rats. At different time points after MSCs or saline infusion, the brain tissue of rats in each group were collected and homogenized in 10 volumes of ice-cold PBS. The IL-1β (A) , IL-6 (B) , TNF-α (C) , IL-10 (D) , MCP-1 (E) , and Rantes (F) levels in the rat's brain tissue lysates were assayed. n = 10 rats per group. * P
    Figure Legend Snippet: MSC administration modulates inflammatory and chemotactic cytokines in the brain tissue of HS rats. At different time points after MSCs or saline infusion, the brain tissue of rats in each group were collected and homogenized in 10 volumes of ice-cold PBS. The IL-1β (A) , IL-6 (B) , TNF-α (C) , IL-10 (D) , MCP-1 (E) , and Rantes (F) levels in the rat's brain tissue lysates were assayed. n = 10 rats per group. * P

    Techniques Used:

    7) Product Images from "Depletion of Regulatory T Lymphocytes Reverses the Imbalance between Pro- and Anti-Tumor Immunities via Enhancing Antigen-Specific T Cell Immune Responses"

    Article Title: Depletion of Regulatory T Lymphocytes Reverses the Imbalance between Pro- and Anti-Tumor Immunities via Enhancing Antigen-Specific T Cell Immune Responses

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0047190

    The concentrations of cytokine profiles in ascites and kinetic changes of various lymphocytes in splenocytes of WF-3-challenged mice treated with PBS or CD25 monoclonal antibody. ( A ) Concentrations of various cytokines in ascites. A1, IL-6; A2, IL-12; A3, TNF-α; A4, IFN-γ; A5, TGF-β. Note : Compared to mice treated with PBS only, the concentrations of cytokines like IL-6, IL-12, TNF-α, and IFN-γ were significantly elevated in mice treated with kinetic low-dose CD25 antibody than those in the other groups. The concentrations of TGF-β significantly decreased in the kinetic low-dose CD25 Ab group compared to the other groups. ( B ) Percentages of various lymphocytes. B1, CD3 + CD4 + help T lymphocytes. Note : The percentages of CD4 + T lymphocytes in the splenocytes were not different between the PBS and CD25 Ab depletion groups on day 14 after WF-3 tumor challenge. However, the kinetic low-dose CD25 Ab depletion group had higher percentages of CD4 + helper T cells than the PBS and the other two sequential CD25 Ab depletion groups after 49 days of tumor challenge. B2, CD3 + CD8 + cytotoxic T lymphocytes. Note : The percentages of CD8 + T cells were not different between the PBS and CD25 Ab depletion groups on 14 days after tumor challenge. The kinetic low-dose CD25 Ab depletion group had higher percentages of CD8 + cytotoxic T cells than the other groups on day 49 after tumor challenge. B3, CD4 + CD25 + Foxp3 + Treg cells. Note : The percentages of Treg cells in the splenocytes were not different among these groups on day 14 after tumor challenge. However, the kinetic low-dose CD25 Ab depletion group had lower percentages of Treg cells than the other groups on day 49. B4, ratios of cytotoxic T lymphocytes/Treg cells. Note : The ratios of CD8 + cytotoxic T cells/Treg cells in the kinetic low-dose CD25 Ab depletion group were highest among the four groups. ( C ) Mesothelin-specific IFN-γ ELISPOT assays of splenocytes in the PBS and CD25 Ab-treated groups. C1, the representative figures of mesothelin-specific IFN-γ ELISPOT assays of splenocytes; C2, bar figures of the numbers of IFN-γ-secreting, mesothelin-specific T lymphocytes in various groups. Note: The kinetic low-dose CD25 Ab depletion group generated the highest numbers of IFN-γ-secreting, mesothelin-specific T lymphocytes compared to the other groups. ( D ) Survivals of mice treated with PBS, neutralizing TGF-β, neutralizing IL-10 or CD25 monoclonal antibodies. D1, Diagrammatic representation of the different treatment protocols of neutralizing TGF-β, neutralizing IL-10 or CD25 monoclonal antibody; D2, Survival curves of mice treated with PBS, neutralizing TGF-β, neutralizing IL-10, neutralizing TGF-β and neutralizing IL-10 or CD25 monoclonal antibody. Note: The mice treated with kinetic low-dose CD25 Ab had the longest survival time after WF-3 tumor challenge when compared to the other mice treated with PBS, neutralizing TGF-β, neutralizing IL-10 or neutralizing TGF-β and neutralizing IL-10 monoclonal antibodies.
    Figure Legend Snippet: The concentrations of cytokine profiles in ascites and kinetic changes of various lymphocytes in splenocytes of WF-3-challenged mice treated with PBS or CD25 monoclonal antibody. ( A ) Concentrations of various cytokines in ascites. A1, IL-6; A2, IL-12; A3, TNF-α; A4, IFN-γ; A5, TGF-β. Note : Compared to mice treated with PBS only, the concentrations of cytokines like IL-6, IL-12, TNF-α, and IFN-γ were significantly elevated in mice treated with kinetic low-dose CD25 antibody than those in the other groups. The concentrations of TGF-β significantly decreased in the kinetic low-dose CD25 Ab group compared to the other groups. ( B ) Percentages of various lymphocytes. B1, CD3 + CD4 + help T lymphocytes. Note : The percentages of CD4 + T lymphocytes in the splenocytes were not different between the PBS and CD25 Ab depletion groups on day 14 after WF-3 tumor challenge. However, the kinetic low-dose CD25 Ab depletion group had higher percentages of CD4 + helper T cells than the PBS and the other two sequential CD25 Ab depletion groups after 49 days of tumor challenge. B2, CD3 + CD8 + cytotoxic T lymphocytes. Note : The percentages of CD8 + T cells were not different between the PBS and CD25 Ab depletion groups on 14 days after tumor challenge. The kinetic low-dose CD25 Ab depletion group had higher percentages of CD8 + cytotoxic T cells than the other groups on day 49 after tumor challenge. B3, CD4 + CD25 + Foxp3 + Treg cells. Note : The percentages of Treg cells in the splenocytes were not different among these groups on day 14 after tumor challenge. However, the kinetic low-dose CD25 Ab depletion group had lower percentages of Treg cells than the other groups on day 49. B4, ratios of cytotoxic T lymphocytes/Treg cells. Note : The ratios of CD8 + cytotoxic T cells/Treg cells in the kinetic low-dose CD25 Ab depletion group were highest among the four groups. ( C ) Mesothelin-specific IFN-γ ELISPOT assays of splenocytes in the PBS and CD25 Ab-treated groups. C1, the representative figures of mesothelin-specific IFN-γ ELISPOT assays of splenocytes; C2, bar figures of the numbers of IFN-γ-secreting, mesothelin-specific T lymphocytes in various groups. Note: The kinetic low-dose CD25 Ab depletion group generated the highest numbers of IFN-γ-secreting, mesothelin-specific T lymphocytes compared to the other groups. ( D ) Survivals of mice treated with PBS, neutralizing TGF-β, neutralizing IL-10 or CD25 monoclonal antibodies. D1, Diagrammatic representation of the different treatment protocols of neutralizing TGF-β, neutralizing IL-10 or CD25 monoclonal antibody; D2, Survival curves of mice treated with PBS, neutralizing TGF-β, neutralizing IL-10, neutralizing TGF-β and neutralizing IL-10 or CD25 monoclonal antibody. Note: The mice treated with kinetic low-dose CD25 Ab had the longest survival time after WF-3 tumor challenge when compared to the other mice treated with PBS, neutralizing TGF-β, neutralizing IL-10 or neutralizing TGF-β and neutralizing IL-10 monoclonal antibodies.

    Techniques Used: Mouse Assay, Enzyme-linked Immunospot, Generated

    Kinetic changes of local immune effector cells in TACs of mice challenged with PBS or WF-3 tumor cells. ( A ) Percentages of various kinds of lymphocytes in TACs of naïve mice and mice of early and advanced diseases. A1, CD4 + helper T lymphocytes; A2, CD8 + cytotoxic T lymphocytes; A3, NK1.1 + natural killer cells; and A4, CD19 + B lymphocytes. Note : For helper and cytotoxic T lymphocytes, the percentages on TACs increased from early to advanced disease. However, the percentages of natural killer cells and lymphocytes in TACs significantly decreased with tumor progression. ( B ) CD223 − CD8 + lymphocytes in TACs of mice. B1: Representative figures of flow cytometric analyses of CD223 − CD8 + and CD223 + CD8 + lymphocytes in TACs. B2: Percentages of CD223 − CD8 + lymphocytes in TACs of mice. Note : Majority of CD8 + cytotoxic T lymphocytes were non-activated as the disease progressed to advanced status. ( C ) The RT-PCR of various cytokines in splenocytes of mice challenged with PBS or WF-3 tumor cells. Note : The expression levels of IL-4, IL-12, TNF-α, and INF-γ decreased but the expression levels of IL-10 increased gradually as the disease progressed. ( D ) The concentrations of various cytokines by ELISA in ascites of mice challenged with PBS or WF-3 tumor cells. D1, IL-12; D2, TNF-α; D3, IFN-γ; D4, IL-6; D5, IL-10; D6, TGF-β. Note : The concentrations of IL-6, IL-10, and TGF-β were elevated but those of IL-12, TNF-α, and IFN-γ decreased as the tumor progressed.
    Figure Legend Snippet: Kinetic changes of local immune effector cells in TACs of mice challenged with PBS or WF-3 tumor cells. ( A ) Percentages of various kinds of lymphocytes in TACs of naïve mice and mice of early and advanced diseases. A1, CD4 + helper T lymphocytes; A2, CD8 + cytotoxic T lymphocytes; A3, NK1.1 + natural killer cells; and A4, CD19 + B lymphocytes. Note : For helper and cytotoxic T lymphocytes, the percentages on TACs increased from early to advanced disease. However, the percentages of natural killer cells and lymphocytes in TACs significantly decreased with tumor progression. ( B ) CD223 − CD8 + lymphocytes in TACs of mice. B1: Representative figures of flow cytometric analyses of CD223 − CD8 + and CD223 + CD8 + lymphocytes in TACs. B2: Percentages of CD223 − CD8 + lymphocytes in TACs of mice. Note : Majority of CD8 + cytotoxic T lymphocytes were non-activated as the disease progressed to advanced status. ( C ) The RT-PCR of various cytokines in splenocytes of mice challenged with PBS or WF-3 tumor cells. Note : The expression levels of IL-4, IL-12, TNF-α, and INF-γ decreased but the expression levels of IL-10 increased gradually as the disease progressed. ( D ) The concentrations of various cytokines by ELISA in ascites of mice challenged with PBS or WF-3 tumor cells. D1, IL-12; D2, TNF-α; D3, IFN-γ; D4, IL-6; D5, IL-10; D6, TGF-β. Note : The concentrations of IL-6, IL-10, and TGF-β were elevated but those of IL-12, TNF-α, and IFN-γ decreased as the tumor progressed.

    Techniques Used: Mouse Assay, Flow Cytometry, Reverse Transcription Polymerase Chain Reaction, Expressing, Enzyme-linked Immunosorbent Assay

    Different expressions of immune components in ascites of early- and advanced-stage ovarian cancer patients. ( A ) Representative figures of flow cytometric analyses of CD4 + helper T cells, CD8 + cytotoxic T lymphocytes, and CD4 + CD25 + regulatory T lymphocytes (Treg cells) in TAL. A1, CD4 + helper T cells; A2, CD8 + cytotoxic T lymphocytes; A3, CD4 + CD25 + Treg cells. ( B ) Percentages of CD4 + helper T lymphocytes, CD8 + cytotoxic T lymphocytes, and CD4 + CD25 + regulatory T lymphocytes (Treg cells) in TAL. B1, CD4 + helper T cells; B2, CD8 + cytotoxic T lymphocytes; B3, CD4 + CD25 + Treg cells. Note : The percentages of CD4 + T cells were significantly higher in patients with advanced stage ovarian cancer. ( C ) Concentrations of various cytokines in ascites of ovarian cancer patients. C1, IL-4; C2, TNF-α; C3, TGF-β; C4, IL-6; C5, IL-10; C6, IFN-γ. Note : The IL-4 and TNF-α concentrations decreased while the TGF-β, IL-6, IL-10, and IFN-γ concentrations increased from early to advanced stage.
    Figure Legend Snippet: Different expressions of immune components in ascites of early- and advanced-stage ovarian cancer patients. ( A ) Representative figures of flow cytometric analyses of CD4 + helper T cells, CD8 + cytotoxic T lymphocytes, and CD4 + CD25 + regulatory T lymphocytes (Treg cells) in TAL. A1, CD4 + helper T cells; A2, CD8 + cytotoxic T lymphocytes; A3, CD4 + CD25 + Treg cells. ( B ) Percentages of CD4 + helper T lymphocytes, CD8 + cytotoxic T lymphocytes, and CD4 + CD25 + regulatory T lymphocytes (Treg cells) in TAL. B1, CD4 + helper T cells; B2, CD8 + cytotoxic T lymphocytes; B3, CD4 + CD25 + Treg cells. Note : The percentages of CD4 + T cells were significantly higher in patients with advanced stage ovarian cancer. ( C ) Concentrations of various cytokines in ascites of ovarian cancer patients. C1, IL-4; C2, TNF-α; C3, TGF-β; C4, IL-6; C5, IL-10; C6, IFN-γ. Note : The IL-4 and TNF-α concentrations decreased while the TGF-β, IL-6, IL-10, and IFN-γ concentrations increased from early to advanced stage.

    Techniques Used: Flow Cytometry

    8) Product Images from "The Impact of Anterior Cruciate Ligament Injury on Lubricin Metabolism and the Effect of Inhibiting TNF-? on Chondroprotection in an Animal Model"

    Article Title: The Impact of Anterior Cruciate Ligament Injury on Lubricin Metabolism and the Effect of Inhibiting TNF-? on Chondroprotection in an Animal Model

    Journal:

    doi: 10.1002/art.24800

    Association of cytokine levels and lubricin expression in ACL transected joints. A Synovial fluid (SF) lavage TNF-α and IL-1β concentrations in ACLT joints at 1 and 4 weeks following transection. There were no detectable levels in the
    Figure Legend Snippet: Association of cytokine levels and lubricin expression in ACL transected joints. A Synovial fluid (SF) lavage TNF-α and IL-1β concentrations in ACLT joints at 1 and 4 weeks following transection. There were no detectable levels in the

    Techniques Used: Expressing

    9) Product Images from "IL-17-dependent Autoimmunity to Collagen Type V in Atherosclerosis"

    Article Title: IL-17-dependent Autoimmunity to Collagen Type V in Atherosclerosis

    Journal: Circulation research

    doi: 10.1161/CIRCRESAHA.110.221069

    ApoE−/− mice on a high fat diet have T and B cell autoimmunity to col(V) ApoE−/− and B6 control mice were fed a high fat diet 15 weeks prior to experimentation. All mice were immunized with TT/DT 2 weeks prior to assay. A, TV-DTH measured T cell responses by splenocytes from ApoE−/− (n=9-21) or B6 control (n=15-21) mice. Responses are against TT/DT, col(V), col(II), col(I), and col(IV). Horizontal bars denote group means. Statistical significance is in comparison to the ApoE−/− col(V) swelling response. B, ApoE−/− mice with a swelling response > 50mm-3 to col(V) (n=7 mice) were selected for analysis. TV-DTH responses were compared between splenocytes injected with col(V) alone or in the presence of neutralizing antibodies to IFN-γ, IL-17, IL-1β, or TNF-α. Statistical significance is in comparison to swelling responses obtained with col(V) alone. C, Serum samples were analyzed for col(V)-specific antibodies (n=10 mice per group). Statistical significance in is comparision to antibody MFI obtained from ApoE−/− mice on a high fat diet. Horizontal bars represent the mean value for each group. *p≤0.05, **p≤0.005, and ***p≤0.0001.
    Figure Legend Snippet: ApoE−/− mice on a high fat diet have T and B cell autoimmunity to col(V) ApoE−/− and B6 control mice were fed a high fat diet 15 weeks prior to experimentation. All mice were immunized with TT/DT 2 weeks prior to assay. A, TV-DTH measured T cell responses by splenocytes from ApoE−/− (n=9-21) or B6 control (n=15-21) mice. Responses are against TT/DT, col(V), col(II), col(I), and col(IV). Horizontal bars denote group means. Statistical significance is in comparison to the ApoE−/− col(V) swelling response. B, ApoE−/− mice with a swelling response > 50mm-3 to col(V) (n=7 mice) were selected for analysis. TV-DTH responses were compared between splenocytes injected with col(V) alone or in the presence of neutralizing antibodies to IFN-γ, IL-17, IL-1β, or TNF-α. Statistical significance is in comparison to swelling responses obtained with col(V) alone. C, Serum samples were analyzed for col(V)-specific antibodies (n=10 mice per group). Statistical significance in is comparision to antibody MFI obtained from ApoE−/− mice on a high fat diet. Horizontal bars represent the mean value for each group. *p≤0.05, **p≤0.005, and ***p≤0.0001.

    Techniques Used: Mouse Assay, Injection

    Patients with coronary artery disease have an autoimmune response to col(V) A, TV-DTH responses by PBMC from age-matched controls or end-stage CAD patients. Responses to EBV recall antigen (positive control), col(V), col(II), or col(I), shown as individual data points, were averaged from duplicate tests. Horizontal bars denote group means. The p value of comparison between CAD and control swelling responses to col(V) is shown. B, Dose-response in TV-DTH assay of PBMC from n=3 CAD patients and n=2 controls to col(V), or to separate α1(V) and α2(V) chains. C, TV-DTH responses to col(V) by whole PBMC, CD4 T cell-depleted PBMC, CD8 T cell-depleted, and CD14 monocyte depleted PBMC for n=4 CAD patients. Data points display mean and standard deviation for n=1-3 depletion experiments per patient. p value shown compares col(V) responses between CD4 T cell depletion or CD14 monocyte depleted and whole PBMC. D, CAD patients (n=3-5) whose TV-DTH responses to col(V) were > 75mm-3 were selected. PBMC were co-injected with col(V) and either IgG isotype control or neutralizing antibodies to IL-2, IL-4, IFN-γ, IL-17, IL-22 (n=4), IL-1β, or TNF-α. Horizontal bars represent the mean value for each group. Responses were compared with IgG isotype control injection. *p≤0.05; **p≤0.01, ***p≤0.0002.
    Figure Legend Snippet: Patients with coronary artery disease have an autoimmune response to col(V) A, TV-DTH responses by PBMC from age-matched controls or end-stage CAD patients. Responses to EBV recall antigen (positive control), col(V), col(II), or col(I), shown as individual data points, were averaged from duplicate tests. Horizontal bars denote group means. The p value of comparison between CAD and control swelling responses to col(V) is shown. B, Dose-response in TV-DTH assay of PBMC from n=3 CAD patients and n=2 controls to col(V), or to separate α1(V) and α2(V) chains. C, TV-DTH responses to col(V) by whole PBMC, CD4 T cell-depleted PBMC, CD8 T cell-depleted, and CD14 monocyte depleted PBMC for n=4 CAD patients. Data points display mean and standard deviation for n=1-3 depletion experiments per patient. p value shown compares col(V) responses between CD4 T cell depletion or CD14 monocyte depleted and whole PBMC. D, CAD patients (n=3-5) whose TV-DTH responses to col(V) were > 75mm-3 were selected. PBMC were co-injected with col(V) and either IgG isotype control or neutralizing antibodies to IL-2, IL-4, IFN-γ, IL-17, IL-22 (n=4), IL-1β, or TNF-α. Horizontal bars represent the mean value for each group. Responses were compared with IgG isotype control injection. *p≤0.05; **p≤0.01, ***p≤0.0002.

    Techniques Used: Positive Control, Standard Deviation, Injection

    10) Product Images from "A Zinc-Dependent Metalloproteinase of Brucella abortus Is Required in the Intracellular Adaptation of Macrophages"

    Article Title: A Zinc-Dependent Metalloproteinase of Brucella abortus Is Required in the Intracellular Adaptation of Macrophages

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2020.01586

    Attenuation of B. abortus by deletion of ZnMP in macrophages. Expression of secondary signals for T cells from B. abortus strain-infected macrophages at MOI 1:10. (A) Production of TNF-α (pg/ml) and (B) production of IL-6 (pg/ml) from macrophages infected with Brucella strains were measured by Sandwich ELISA at 6 and 24 h pi with standard curves using TNF-α and IL-6 recombinant proteins. (C,E) Expression of co-stimulatory proteins CD80 and CD86 from infected macrophages measured by flow cytometry at 6 h pi (blue) and 24 h pi (red). Fluorescence minus one (FMO) was used as a negative control. (D,F) show the mean ± standard deviation of the mean fluorescence intensity (MFI) of CD80 and CD86. Data represent unstimulated (PBS-treated) macrophages, B. abortus 2308-infected macrophages, B. abortus Δ270-infected macrophages and B. abortus 2308i (inactivated strain) stimulated macrophages. Results were expressed as mean ± standard deviation. Values of P
    Figure Legend Snippet: Attenuation of B. abortus by deletion of ZnMP in macrophages. Expression of secondary signals for T cells from B. abortus strain-infected macrophages at MOI 1:10. (A) Production of TNF-α (pg/ml) and (B) production of IL-6 (pg/ml) from macrophages infected with Brucella strains were measured by Sandwich ELISA at 6 and 24 h pi with standard curves using TNF-α and IL-6 recombinant proteins. (C,E) Expression of co-stimulatory proteins CD80 and CD86 from infected macrophages measured by flow cytometry at 6 h pi (blue) and 24 h pi (red). Fluorescence minus one (FMO) was used as a negative control. (D,F) show the mean ± standard deviation of the mean fluorescence intensity (MFI) of CD80 and CD86. Data represent unstimulated (PBS-treated) macrophages, B. abortus 2308-infected macrophages, B. abortus Δ270-infected macrophages and B. abortus 2308i (inactivated strain) stimulated macrophages. Results were expressed as mean ± standard deviation. Values of P

    Techniques Used: Expressing, Infection, Sandwich ELISA, Recombinant, Flow Cytometry, Fluorescence, Negative Control, Standard Deviation

    11) Product Images from "The α2 Na+/K+-ATPase isoform mediates LPS-induced neuroinflammation"

    Article Title: The α2 Na+/K+-ATPase isoform mediates LPS-induced neuroinflammation

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-71027-5

    The levels of cytokines are reduced in the hippocampus and hypothalamus of α 2 +/G301R mice. α 2 Na + /K + -ATPase haploinsufficiency decreases lipopolysaccharide (LPS)-induced hypothalamic (blue bars) and hippocampal (red bars) cytokine transcription. ( a – f ) TaqMan quantitative PCR analysis of TNF-α ( a , d ); IL-1β ( b , e ); and IL-6 ( c , f ) gene expression relative to the expression of β - actin, used as endogenous reference gene. LPS induced significant increases in the levels of TNF-α in the hypothalamus of both α 2 +/+ ( n = 3), and α 2 +/G301R ( n = 4), mice. LPS induced significant increases in the levels IL-1β and IL-6 in the hypothalamus (blue bars) of α 2 +/+ mice ( n = 3), but not in α 2 +/G301R ( n = 3) mice. In the hippocampus (red bars), LPS induced significant increases in the levels of TNF-α, IL-1β and IL-6 of α 2 +/+ mice ( n = 3), but not in α 2 +/G301R ( n = 4) mice. All data are presented as the mean ± SEM. *p
    Figure Legend Snippet: The levels of cytokines are reduced in the hippocampus and hypothalamus of α 2 +/G301R mice. α 2 Na + /K + -ATPase haploinsufficiency decreases lipopolysaccharide (LPS)-induced hypothalamic (blue bars) and hippocampal (red bars) cytokine transcription. ( a – f ) TaqMan quantitative PCR analysis of TNF-α ( a , d ); IL-1β ( b , e ); and IL-6 ( c , f ) gene expression relative to the expression of β - actin, used as endogenous reference gene. LPS induced significant increases in the levels of TNF-α in the hypothalamus of both α 2 +/+ ( n = 3), and α 2 +/G301R ( n = 4), mice. LPS induced significant increases in the levels IL-1β and IL-6 in the hypothalamus (blue bars) of α 2 +/+ mice ( n = 3), but not in α 2 +/G301R ( n = 3) mice. In the hippocampus (red bars), LPS induced significant increases in the levels of TNF-α, IL-1β and IL-6 of α 2 +/+ mice ( n = 3), but not in α 2 +/G301R ( n = 4) mice. All data are presented as the mean ± SEM. *p

    Techniques Used: Mouse Assay, Real-time Polymerase Chain Reaction, Expressing

    The α 2 Na + /K + -ATPase isoform is not involved macrophage cytokine production in response to LPS. BMDMs derived from bone marrow cells from the femurs and tibias of α 2 +/+ and α 2 +/G301R mice were cultured in L929 cell supernatant for 6 days. After 6 days, the BMDMs were treated with LPS (100 ng/mL) or PBS (CTR). ( a – d ) The levels of TNF-α were measured in the supernatant 1 h ( a ), 2 h ( b ), 4 h ( c ), and 6 h ( d ) after LPS treatment. ( e ) α 2 Isoform levels were measured by Western blotting in bone marrow derived macrophages from α 2 +/+ and α 2 +/G301R mice at the indicated times during differentiation. β-Actin was used as a loading control. A protein extract from the central nervous system (CNS) was loaded as a positive control. ( f ) Densitometric analysis from day 0 (arbitrary units, A.U.) showing that no obvious differences existed in the α 2 protein levels in the BMDMs derived from the α 2 +/+ and α 2 +/G301R mice. The data are presented as the mean ± SEM from two individual experiments. The data are presented as the mean ± SEM. *p
    Figure Legend Snippet: The α 2 Na + /K + -ATPase isoform is not involved macrophage cytokine production in response to LPS. BMDMs derived from bone marrow cells from the femurs and tibias of α 2 +/+ and α 2 +/G301R mice were cultured in L929 cell supernatant for 6 days. After 6 days, the BMDMs were treated with LPS (100 ng/mL) or PBS (CTR). ( a – d ) The levels of TNF-α were measured in the supernatant 1 h ( a ), 2 h ( b ), 4 h ( c ), and 6 h ( d ) after LPS treatment. ( e ) α 2 Isoform levels were measured by Western blotting in bone marrow derived macrophages from α 2 +/+ and α 2 +/G301R mice at the indicated times during differentiation. β-Actin was used as a loading control. A protein extract from the central nervous system (CNS) was loaded as a positive control. ( f ) Densitometric analysis from day 0 (arbitrary units, A.U.) showing that no obvious differences existed in the α 2 protein levels in the BMDMs derived from the α 2 +/+ and α 2 +/G301R mice. The data are presented as the mean ± SEM from two individual experiments. The data are presented as the mean ± SEM. *p

    Techniques Used: Derivative Assay, Mouse Assay, Cell Culture, Western Blot, Positive Control

    The level of Tlr4 is reduced in the hippocampus and hypothalamus of α 2 +/G301R mice. The α 2 +/G301R mice exhibited reduced Tlr4 and Tnf-α mRNA expression in astrocytes isolated from the hypothalamus (blue bars) and hippocampus (red bars) compared with that in the α 2 +/+ mice after PBS treatment. ( a , b ) qPCR analysis of Tlr4 relative to β-actin expression in the hypothalamus ( a ) and hippocampus ( b ) showed a significant reduction of Trl4 in these brain areas, notably after LPS treatment, between the α 2 +/+ ( n = 3) and α 2 +/G301R ( n = 3) animals. The qPCR analysis of Tnf-α relative to β-actin expression in the hypothalamus ( c ) and hippocampus ( d ) showed that a significant decrease in Tnf-α mRNA expression was observed in astrocytes from α 2 +/G301R mice ( n = 3) after LPS treatment compared with α 2 +/+ astrocytes ( n = 3). All data are presented as the mean ± SEM. *p
    Figure Legend Snippet: The level of Tlr4 is reduced in the hippocampus and hypothalamus of α 2 +/G301R mice. The α 2 +/G301R mice exhibited reduced Tlr4 and Tnf-α mRNA expression in astrocytes isolated from the hypothalamus (blue bars) and hippocampus (red bars) compared with that in the α 2 +/+ mice after PBS treatment. ( a , b ) qPCR analysis of Tlr4 relative to β-actin expression in the hypothalamus ( a ) and hippocampus ( b ) showed a significant reduction of Trl4 in these brain areas, notably after LPS treatment, between the α 2 +/+ ( n = 3) and α 2 +/G301R ( n = 3) animals. The qPCR analysis of Tnf-α relative to β-actin expression in the hypothalamus ( c ) and hippocampus ( d ) showed that a significant decrease in Tnf-α mRNA expression was observed in astrocytes from α 2 +/G301R mice ( n = 3) after LPS treatment compared with α 2 +/+ astrocytes ( n = 3). All data are presented as the mean ± SEM. *p

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

    α 2 +/G301R mice display decreased sickness behaviour. α 2 +/G301R mice experience hypothermia, and systemic proinflammatory cytokine levels compared to α 2 +/+ mice following LPS administration (500 µg/kg, intraperitoneally (IP) injected). ( a ) Example of sickness behaviour 4 h after LPS administration. As illustrated, the α 2 +/+ mice displayed sickness behaviour, less locomotion, and, briefly, hypothermia, in contrast to the α 2 +/G301R mice that displayed less sickness behaviour after LPS administration. ( b ) The differences in body temperature 4 h after LPS administration compared to baseline (t = 0) in the α 2 +/+ and α 2 +/G301R animals treated with PBS or LPS, with a significant different after LPS treatment. ( c – e ) The α 2 +/G301R mice and their WT littermates (α 2 +/+ ) were injected with saline or LPS (500 µg/kg), and the levels of TNF-α ( c ) ( n = 12 for the α 2 +/+ mice and n = 14 for α 2 +/G301R mice), IL-1β ( d ) [α 2 +/+ ( n = 6) and α 2 +/G301R mice ( n = 7)] and IL-6 ( e ) [α 2 +/+ ( n = 6) and α 2 +/G301R ( n = 7)] were measured in the serum 4 h after saline/LPS treatment. The data are presented as the mean ± SEM. *p
    Figure Legend Snippet: α 2 +/G301R mice display decreased sickness behaviour. α 2 +/G301R mice experience hypothermia, and systemic proinflammatory cytokine levels compared to α 2 +/+ mice following LPS administration (500 µg/kg, intraperitoneally (IP) injected). ( a ) Example of sickness behaviour 4 h after LPS administration. As illustrated, the α 2 +/+ mice displayed sickness behaviour, less locomotion, and, briefly, hypothermia, in contrast to the α 2 +/G301R mice that displayed less sickness behaviour after LPS administration. ( b ) The differences in body temperature 4 h after LPS administration compared to baseline (t = 0) in the α 2 +/+ and α 2 +/G301R animals treated with PBS or LPS, with a significant different after LPS treatment. ( c – e ) The α 2 +/G301R mice and their WT littermates (α 2 +/+ ) were injected with saline or LPS (500 µg/kg), and the levels of TNF-α ( c ) ( n = 12 for the α 2 +/+ mice and n = 14 for α 2 +/G301R mice), IL-1β ( d ) [α 2 +/+ ( n = 6) and α 2 +/G301R mice ( n = 7)] and IL-6 ( e ) [α 2 +/+ ( n = 6) and α 2 +/G301R ( n = 7)] were measured in the serum 4 h after saline/LPS treatment. The data are presented as the mean ± SEM. *p

    Techniques Used: Mouse Assay, Injection

    12) Product Images from "Dectin-1 Facilitates IL-18 Production for the Generation of Protective Antibodies Against Candida albicans"

    Article Title: Dectin-1 Facilitates IL-18 Production for the Generation of Protective Antibodies Against Candida albicans

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2020.01648

    T H 2 cell response is involved in gpi7 mutant vaccination induced immunoprotection against candidiasis in mice. Gpi7 mutant-vaccinated C57BL/6 mice were systemically infected with C. albicans SC5314 (5 × 10 5 CFU per mouse). (A) ELISA results for cytokines TNF-α, IL-6 in the spleen and serum of gpi7 mutant-vaccinated mice at day 2 post-infection ( n = 5 per group). (B) ELISA results for cytokines IL-4 and IL-13 in the spleen and serum of gpi7 mutant-vaccinated mice at day 2 post-infection ( n = 5 per group). (C) Flow cytometry analysis for CD4 + IL-4 + T cells in the spleen of gpi7 mutant-vaccinated mice at day 2 post-infection. Data are representative images of 5 mice. (D) IL-4 and IL-13 levels in gpi7 mutant-vaccinated mice given intravenous injection of control IgG and neutralizing anti-CD4 antibody (200 μg per mouse) twice ( n = 5 per group). ** P
    Figure Legend Snippet: T H 2 cell response is involved in gpi7 mutant vaccination induced immunoprotection against candidiasis in mice. Gpi7 mutant-vaccinated C57BL/6 mice were systemically infected with C. albicans SC5314 (5 × 10 5 CFU per mouse). (A) ELISA results for cytokines TNF-α, IL-6 in the spleen and serum of gpi7 mutant-vaccinated mice at day 2 post-infection ( n = 5 per group). (B) ELISA results for cytokines IL-4 and IL-13 in the spleen and serum of gpi7 mutant-vaccinated mice at day 2 post-infection ( n = 5 per group). (C) Flow cytometry analysis for CD4 + IL-4 + T cells in the spleen of gpi7 mutant-vaccinated mice at day 2 post-infection. Data are representative images of 5 mice. (D) IL-4 and IL-13 levels in gpi7 mutant-vaccinated mice given intravenous injection of control IgG and neutralizing anti-CD4 antibody (200 μg per mouse) twice ( n = 5 per group). ** P

    Techniques Used: Mutagenesis, Mouse Assay, Infection, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Injection

    13) Product Images from "Phytosterol esters attenuate hepatic steatosis in rats with non-alcoholic fatty liver disease rats fed a high-fat diet"

    Article Title: Phytosterol esters attenuate hepatic steatosis in rats with non-alcoholic fatty liver disease rats fed a high-fat diet

    Journal: Scientific Reports

    doi: 10.1038/srep41604

    Gene expression in hepatic tissues in the different groups. The mRNA and protein expression levels in hepatic tissues in the HF and three PSEs intervention groups were compared with those in the NC group. The PSEs treatments at the three doses resulted in significant down-regulation of TGF-β1, TGF-β2, TNF-α, and UCP-2 expression and slight up-regulation of ELOVL-2 expression. The effects of PSEs on PPAR-α, PPAR-γ and LXR-α expression differed. The data are expressed as the mean ± standard deviation. Δ P
    Figure Legend Snippet: Gene expression in hepatic tissues in the different groups. The mRNA and protein expression levels in hepatic tissues in the HF and three PSEs intervention groups were compared with those in the NC group. The PSEs treatments at the three doses resulted in significant down-regulation of TGF-β1, TGF-β2, TNF-α, and UCP-2 expression and slight up-regulation of ELOVL-2 expression. The effects of PSEs on PPAR-α, PPAR-γ and LXR-α expression differed. The data are expressed as the mean ± standard deviation. Δ P

    Techniques Used: Expressing, Standard Deviation

    14) Product Images from "Neuroprotective Effect of Fagopyrum dibotrys Extract against Alzheimer's Disease"

    Article Title: Neuroprotective Effect of Fagopyrum dibotrys Extract against Alzheimer's Disease

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    doi: 10.1155/2017/3294586

    FDE attenuates inflammation in APP/PS1 transgenic mice. (a–c) Representative images of staining in APP/PS1. No obvious microgliosis was observed in the brain of FDE treatment transgenic mice. (c) Comparison of CD45 area fraction in neocortex among groups. (d) Quantification of IL-1 β , IL-6, TNF- α , and IFN- γ in plasma. Denote p
    Figure Legend Snippet: FDE attenuates inflammation in APP/PS1 transgenic mice. (a–c) Representative images of staining in APP/PS1. No obvious microgliosis was observed in the brain of FDE treatment transgenic mice. (c) Comparison of CD45 area fraction in neocortex among groups. (d) Quantification of IL-1 β , IL-6, TNF- α , and IFN- γ in plasma. Denote p

    Techniques Used: Transgenic Assay, Mouse Assay, Staining

    15) Product Images from "Pre-Treatment of Recombinant Mouse MFG-E8 Downregulates LPS-Induced TNF-? Production in Macrophages via STAT3-Mediated SOCS3 Activation"

    Article Title: Pre-Treatment of Recombinant Mouse MFG-E8 Downregulates LPS-Induced TNF-? Production in Macrophages via STAT3-Mediated SOCS3 Activation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0027685

    Hypothesis scheme. In macrophage cells, LPS triggers TLR4 pathway to activate NF-κB signals by promoting IκB degradation and nuclear translocation of p50 and RelA /p65 to enhance the target gene e.g., TNF-α transcription. In our current study, we demonstrated a non-canonical signaling where LPS may activate the phosphorylation of a ubiquitous transcription factor, STAT3 which in turn can further augment the down-stream signaling for TNF-α expression, since blocking STAT3 activation by Static dramatically reduced the LPS induced TNF-α production. In the biological system, the versatile function of STAT3 enabled it to generate both the inflammatory and anti-inflammatory signal transduction efficiently. Utilizing the STAT3 pathway, MFG-E8 can directly exert its anti-inflammatory effects towards downregulating the LPS-induced TNF-α production in macrophages via activating SOCS3. MFG-E8-induced SOCS3 may in turn target the NF-κB p65 component for ubiquitination and act as a negative regulator of LPS-mediated TLR4 signaling via NF-κB for TNF-α production.
    Figure Legend Snippet: Hypothesis scheme. In macrophage cells, LPS triggers TLR4 pathway to activate NF-κB signals by promoting IκB degradation and nuclear translocation of p50 and RelA /p65 to enhance the target gene e.g., TNF-α transcription. In our current study, we demonstrated a non-canonical signaling where LPS may activate the phosphorylation of a ubiquitous transcription factor, STAT3 which in turn can further augment the down-stream signaling for TNF-α expression, since blocking STAT3 activation by Static dramatically reduced the LPS induced TNF-α production. In the biological system, the versatile function of STAT3 enabled it to generate both the inflammatory and anti-inflammatory signal transduction efficiently. Utilizing the STAT3 pathway, MFG-E8 can directly exert its anti-inflammatory effects towards downregulating the LPS-induced TNF-α production in macrophages via activating SOCS3. MFG-E8-induced SOCS3 may in turn target the NF-κB p65 component for ubiquitination and act as a negative regulator of LPS-mediated TLR4 signaling via NF-κB for TNF-α production.

    Techniques Used: Translocation Assay, Expressing, Blocking Assay, Activation Assay, Transduction, Activated Clotting Time Assay

    Effect of rmMFG-E8 on TNF-α production by LPS treated macrophages. (A, B) RAW264.7 cells (1×10 6 cells) and (C, D) peritoneal macrophages from WT and MFG-E8 −/− mice (0.5×10 6 cells) were plated in 24-well cell culture plates. After pre-incubation with rmMFG-E8 (500 ng/mL) for 2 h, the cells were then stimulated by LPS (10 ng/mL) for 2 h and the expression of TNF-α mRNA (A, C) was checked by real-time PCR. To assess the protein levels of TNF-α (B, D), RAW264.7 cells and mouse peritoneal macrophages were stimulated with LPS for 4 and 24 h, respectively and then ELISA was performed from the culture supernatants. (E) Dose-dependent effects of rmMFG-E8 for inhibiting the LPS-induced TNF-α production in murine peritoneal macrophages (0.5×10 6 cells) cultured in 24-well cell culture plates. (F) Peritoneal macrophages from WT and MFG-E8 −/− mice (0.5×10 6 cells) were plated in 24-well cell culture plates. After pre-incubation with rmMFG-E8 (500 ng/mL) for 2 h, the cells were stimulated by LPS for 24 h, followed by the measurement of IL-1β by ELISA. Data are expressed as means ± SE (n = 3 independent experiments) and compared by one-way ANOVA and SNK method: * P
    Figure Legend Snippet: Effect of rmMFG-E8 on TNF-α production by LPS treated macrophages. (A, B) RAW264.7 cells (1×10 6 cells) and (C, D) peritoneal macrophages from WT and MFG-E8 −/− mice (0.5×10 6 cells) were plated in 24-well cell culture plates. After pre-incubation with rmMFG-E8 (500 ng/mL) for 2 h, the cells were then stimulated by LPS (10 ng/mL) for 2 h and the expression of TNF-α mRNA (A, C) was checked by real-time PCR. To assess the protein levels of TNF-α (B, D), RAW264.7 cells and mouse peritoneal macrophages were stimulated with LPS for 4 and 24 h, respectively and then ELISA was performed from the culture supernatants. (E) Dose-dependent effects of rmMFG-E8 for inhibiting the LPS-induced TNF-α production in murine peritoneal macrophages (0.5×10 6 cells) cultured in 24-well cell culture plates. (F) Peritoneal macrophages from WT and MFG-E8 −/− mice (0.5×10 6 cells) were plated in 24-well cell culture plates. After pre-incubation with rmMFG-E8 (500 ng/mL) for 2 h, the cells were stimulated by LPS for 24 h, followed by the measurement of IL-1β by ELISA. Data are expressed as means ± SE (n = 3 independent experiments) and compared by one-way ANOVA and SNK method: * P

    Techniques Used: Mouse Assay, Cell Culture, Incubation, Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

    In vivo effects of rmMFG-E8 in controlling TNF-α production during endotoxemia. (A) WT mice were injected with rmMFG-E8 (0.4 µg/20 g BW) via i.p. for 2 h and then endotoxemia was generated by an overdose of i.p LPS (5 mg/kg BW) administration. Similarly, in another experiment, WT and MFG-E8 −/− mice were treated with LPS (5 mg/kg BW) via i.p.. After 4 h, mice were euthanized, blood was collected and TNF-α measured by ELISA. (B, C) WT mice were treated with rmMFG-E8 (0.4 µg/20 g BW) for 2 h, followed by LPS (5 mg/kg BW) challenge. WT and MFG-E8 −/− mice were injected with LPS (5 mg/kg BW) i.p.. After 2 h total RNA and after 4 h proteins were extracted from splenic tissues for TNF-α expression using real-time PCR and ELISA, respectively. All data are expressed as means ± SE (n = 3 independent experiments) and compared by one-way ANOVA and SNK method: * P
    Figure Legend Snippet: In vivo effects of rmMFG-E8 in controlling TNF-α production during endotoxemia. (A) WT mice were injected with rmMFG-E8 (0.4 µg/20 g BW) via i.p. for 2 h and then endotoxemia was generated by an overdose of i.p LPS (5 mg/kg BW) administration. Similarly, in another experiment, WT and MFG-E8 −/− mice were treated with LPS (5 mg/kg BW) via i.p.. After 4 h, mice were euthanized, blood was collected and TNF-α measured by ELISA. (B, C) WT mice were treated with rmMFG-E8 (0.4 µg/20 g BW) for 2 h, followed by LPS (5 mg/kg BW) challenge. WT and MFG-E8 −/− mice were injected with LPS (5 mg/kg BW) i.p.. After 2 h total RNA and after 4 h proteins were extracted from splenic tissues for TNF-α expression using real-time PCR and ELISA, respectively. All data are expressed as means ± SE (n = 3 independent experiments) and compared by one-way ANOVA and SNK method: * P

    Techniques Used: In Vivo, Mouse Assay, Injection, Generated, Enzyme-linked Immunosorbent Assay, Expressing, Real-time Polymerase Chain Reaction

    16) Product Images from "Nipah Virus Infection of Immature Dendritic Cells Increases Its Transendothelial Migration Across Human Brain Microvascular Endothelial Cells"

    Article Title: Nipah Virus Infection of Immature Dendritic Cells Increases Its Transendothelial Migration Across Human Brain Microvascular Endothelial Cells

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2018.02747

    Transendothelial migration of iDC, pMO, and THP-1, either mock-infected or infected with NiV, across untreated and TNF-α-treated HBMEC and HUVEC: (A) migration was presented as the percentage of migrated cells [represented on the y-axis as migrated cells (%)], and (B) permeability of endothelial monolayers after transendothelial migration as measured by the flux of 70 kDa fluorescein-dextran (represented on the y-axis as fluorescence units). Values are the average of three biological replicates and error bars indicate standard deviation ( ∗ p
    Figure Legend Snippet: Transendothelial migration of iDC, pMO, and THP-1, either mock-infected or infected with NiV, across untreated and TNF-α-treated HBMEC and HUVEC: (A) migration was presented as the percentage of migrated cells [represented on the y-axis as migrated cells (%)], and (B) permeability of endothelial monolayers after transendothelial migration as measured by the flux of 70 kDa fluorescein-dextran (represented on the y-axis as fluorescence units). Values are the average of three biological replicates and error bars indicate standard deviation ( ∗ p

    Techniques Used: Migration, Infection, Permeability, Fluorescence, Standard Deviation

    17) Product Images from "The time-course and RNA interference of TNF-α, IL-6, and IL-1β expression on neuropathic pain induced by L5 spinal nerve transection in rats"

    Article Title: The time-course and RNA interference of TNF-α, IL-6, and IL-1β expression on neuropathic pain induced by L5 spinal nerve transection in rats

    Journal: Korean Journal of Anesthesiology

    doi: 10.4097/kjae.2015.68.2.159

    Percentage levels of TNF-α, IL-6 and IL-1β in the rats undergoing L5 spinal nerve transection (SNT) after the administration of the control small interfering RNA (siRNA) (CON group). Data are expressed as the median (range). Each cytokine OD level is normalized to the median OD value at 0 h. The L5 spinal nerve cord samples are harvested on 0, 4, 8, 12 h and 1, 2, 4 and 6 d after L5 SNT to detect these cytokine levels by standard enzyme-linked immunosorbent assay (ELISA). The median OD levels of TNF-α, IL-6 and IL-1β in the spinal cord at 7 d after intrathecal catheter implantation without administration of siRNA or a SNT operation are used as t o values. * P
    Figure Legend Snippet: Percentage levels of TNF-α, IL-6 and IL-1β in the rats undergoing L5 spinal nerve transection (SNT) after the administration of the control small interfering RNA (siRNA) (CON group). Data are expressed as the median (range). Each cytokine OD level is normalized to the median OD value at 0 h. The L5 spinal nerve cord samples are harvested on 0, 4, 8, 12 h and 1, 2, 4 and 6 d after L5 SNT to detect these cytokine levels by standard enzyme-linked immunosorbent assay (ELISA). The median OD levels of TNF-α, IL-6 and IL-1β in the spinal cord at 7 d after intrathecal catheter implantation without administration of siRNA or a SNT operation are used as t o values. * P

    Techniques Used: Small Interfering RNA, Enzyme-linked Immunosorbent Assay

    Schematic representation of the experimental procedure used in this study. siRNA: small interfering RNA, CON group: rats allocated to receive control siRNA with a scrambled nucleotide sequence, COCK group: rats allocated to receive the cocktail of siRNAs together targeting TNF-α, IL-6 and IL-1b. In the CON group, cytokine mRNA expressions (n = 7) and the activation of glial cells (n = 3) are determined 7 d after intrathecal catheter implantation without any administration of control siRNA or SNT operation. These data are used as baseline values of cytokine mRNA expression and the activation of glial cells.
    Figure Legend Snippet: Schematic representation of the experimental procedure used in this study. siRNA: small interfering RNA, CON group: rats allocated to receive control siRNA with a scrambled nucleotide sequence, COCK group: rats allocated to receive the cocktail of siRNAs together targeting TNF-α, IL-6 and IL-1b. In the CON group, cytokine mRNA expressions (n = 7) and the activation of glial cells (n = 3) are determined 7 d after intrathecal catheter implantation without any administration of control siRNA or SNT operation. These data are used as baseline values of cytokine mRNA expression and the activation of glial cells.

    Techniques Used: Small Interfering RNA, Sequencing, Activation Assay, Expressing

    The time course of mechanical allodynia (A) and hyperalgesia (B) in the ipsilateral hind paw of rats undergoing L5 spinal nerve transection (SNT) after the administration of control siRNA (CON group) or a cocktail of small interfering RNAs (siRNA) targeting TNF-α, IL-6 and IL1-β (COCK group). The data on the rats surviving for 6 d after SNT are expressed as mean ± SE. -1: 1 d prior to SNT, 0: the day of L5 SNT, 1, 2, 4 and 6: 1, 2, 4 and 6 d after L5 SNT. * P
    Figure Legend Snippet: The time course of mechanical allodynia (A) and hyperalgesia (B) in the ipsilateral hind paw of rats undergoing L5 spinal nerve transection (SNT) after the administration of control siRNA (CON group) or a cocktail of small interfering RNAs (siRNA) targeting TNF-α, IL-6 and IL1-β (COCK group). The data on the rats surviving for 6 d after SNT are expressed as mean ± SE. -1: 1 d prior to SNT, 0: the day of L5 SNT, 1, 2, 4 and 6: 1, 2, 4 and 6 d after L5 SNT. * P

    Techniques Used:

    mRNA expression of TNF-α, IL-6, and IL-1β in rats undergoing L5 spinal nerve transection (SNT) (after the administration of control small interfering RNA (siRNA) (CON group). Tissue samples are acquired at 4, 8 and 12 h and 1, 2, 4 and 6 d (n = 3 per each time point) after L5 SNT, and mRNAs for the cytokines are isolated and quantified by reverse transcription polymerase chain reaction (RT-PCR). Cyclophilin is used as a house keeping gene. BASE: 7 d after intrathecal catheter implantation without administration of control siRNA (n = 3). Cyc: Cyclophilin.
    Figure Legend Snippet: mRNA expression of TNF-α, IL-6, and IL-1β in rats undergoing L5 spinal nerve transection (SNT) (after the administration of control small interfering RNA (siRNA) (CON group). Tissue samples are acquired at 4, 8 and 12 h and 1, 2, 4 and 6 d (n = 3 per each time point) after L5 SNT, and mRNAs for the cytokines are isolated and quantified by reverse transcription polymerase chain reaction (RT-PCR). Cyclophilin is used as a house keeping gene. BASE: 7 d after intrathecal catheter implantation without administration of control siRNA (n = 3). Cyc: Cyclophilin.

    Techniques Used: Expressing, Small Interfering RNA, Isolation, Reverse Transcription Polymerase Chain Reaction

    mRNA expression of TNF-α, IL-6, and IL-1β in rats undergoing L5 spinal nerve transection (SNT) after the administration of the cocktail of small interfering RNA (siRNA) targeting TNF-α, IL-6 and IL-1β (COCK group). See legend to Fig. 3 for further details. The mRNA levels for TNF-α, IL-6 and IL-1β at 4 h after SNT are not determined because of a technical error which occurred during the analysis process.
    Figure Legend Snippet: mRNA expression of TNF-α, IL-6, and IL-1β in rats undergoing L5 spinal nerve transection (SNT) after the administration of the cocktail of small interfering RNA (siRNA) targeting TNF-α, IL-6 and IL-1β (COCK group). See legend to Fig. 3 for further details. The mRNA levels for TNF-α, IL-6 and IL-1β at 4 h after SNT are not determined because of a technical error which occurred during the analysis process.

    Techniques Used: Expressing, Small Interfering RNA

    mRNA expression of TNF-α, IL-6 and IL-1β in rats undergoing L5 spinal nerve transection (SNT) and administration of control small interfering RNA (siRNA) (CON group) or the cocktail of siRNA targeting TNF-α, IL-6 and IL-1β (COCK group). Data are expressed as medians (ranges). Cytokine mRNA expression levels are normalized to cyclophilin. 0 h: TNF-α, IL-6 and IL-1β mRNA levels in the spinal cord at 7 d after intrathecal catheter implantation without any administration of control siRNA or the SNT operation are used as the values at time 0 h (n = 3). The cytokine levels at 4 h in the COCK group are not determined because of a technical error during the analysis. * P
    Figure Legend Snippet: mRNA expression of TNF-α, IL-6 and IL-1β in rats undergoing L5 spinal nerve transection (SNT) and administration of control small interfering RNA (siRNA) (CON group) or the cocktail of siRNA targeting TNF-α, IL-6 and IL-1β (COCK group). Data are expressed as medians (ranges). Cytokine mRNA expression levels are normalized to cyclophilin. 0 h: TNF-α, IL-6 and IL-1β mRNA levels in the spinal cord at 7 d after intrathecal catheter implantation without any administration of control siRNA or the SNT operation are used as the values at time 0 h (n = 3). The cytokine levels at 4 h in the COCK group are not determined because of a technical error during the analysis. * P

    Techniques Used: Expressing, Small Interfering RNA

    18) Product Images from "Characterizing the BCG Induced Macrophage and Neutrophil Mechanisms for Defense Against Mycobacterium tuberculosis"

    Article Title: Characterizing the BCG Induced Macrophage and Neutrophil Mechanisms for Defense Against Mycobacterium tuberculosis

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2020.01202

    C57BL/6 mice were vaccinated with either live BCG or γ-irradiated BCG (irradiated BCG) subcutaneously or intravenously. γ-irradiated BCG was unable to stimulate a reduction in mycobacterial burden compared to live BCG which was able to reduce mycobacterial burden by 1 Log 10 (A) . N = 5–6 mice per group, * p ≤ 0.05. The reporter RAW-Blue® cell line to assess NF-κB and AP-1 activation was used to assess the ability of either live BCG or γ-irradiated BCG to stimulate NF-kB and AP-1 signaling pathways. The activation of NF-kB and AP-1 signaling pathways was similar, regardless of whether the BCG was viable or non-replicating (B) . Cytokine mRNA expression was examined in C57BL/6-derived BMDM, stimulated with BCG for 24 h, using the RT2 profiler™ PCR array for mouse cytokines and chemokines (C) . Supernatants were also collected from these cultures and assessed for the production of protein cytokines TNF-α, IL-1β, IL-6, and IL-10 (D) . The in vivo data is a representative of multiple iterations with N = 5 mice per group. Data are representative of two in vitro studies.
    Figure Legend Snippet: C57BL/6 mice were vaccinated with either live BCG or γ-irradiated BCG (irradiated BCG) subcutaneously or intravenously. γ-irradiated BCG was unable to stimulate a reduction in mycobacterial burden compared to live BCG which was able to reduce mycobacterial burden by 1 Log 10 (A) . N = 5–6 mice per group, * p ≤ 0.05. The reporter RAW-Blue® cell line to assess NF-κB and AP-1 activation was used to assess the ability of either live BCG or γ-irradiated BCG to stimulate NF-kB and AP-1 signaling pathways. The activation of NF-kB and AP-1 signaling pathways was similar, regardless of whether the BCG was viable or non-replicating (B) . Cytokine mRNA expression was examined in C57BL/6-derived BMDM, stimulated with BCG for 24 h, using the RT2 profiler™ PCR array for mouse cytokines and chemokines (C) . Supernatants were also collected from these cultures and assessed for the production of protein cytokines TNF-α, IL-1β, IL-6, and IL-10 (D) . The in vivo data is a representative of multiple iterations with N = 5 mice per group. Data are representative of two in vitro studies.

    Techniques Used: Mouse Assay, Irradiation, Activation Assay, Expressing, Derivative Assay, Polymerase Chain Reaction, In Vivo, In Vitro

    C57BL/6 CD4, CD8, and TNF-α knockout mice were vaccinated subcutaneously with 5 × 10 4 CFU BCG, rested for 30 days and then infected with a low dose aerosol of M. tuberculosis H37Rv. CFU were determined at days 30 and 60 post-infection for the CD4 −/− and CD8 −/− mice and day 30 for the TNF-α KO mice (A) . C57BL/6 mice vaccinated with 5 × 10 4 CFU BCG Pasteur 7 days before infection and the CFU determined 30 days after infection in lungs and spleens (B) . The concentration of and TNF-α, IFN-γ, and IL-2 as determined by cytometric bead array (CBA) at day 30 post-infection when mice were vaccinated 7 and 14 days infection (C) . IFNγ producing cells present in spleen as determined by ELISpot after stimulation with H37Rv Culture filtrate protein (CFP) after infection when mice are vaccinated 7 and 14 days before infection (D) . Experiments were performed with N = 4–5 mice per group and are representative of multiple iterations. * p ≤ 0.05 ** p ≤ 0.01, *** p ≤ 0.001, n.s., not significant.
    Figure Legend Snippet: C57BL/6 CD4, CD8, and TNF-α knockout mice were vaccinated subcutaneously with 5 × 10 4 CFU BCG, rested for 30 days and then infected with a low dose aerosol of M. tuberculosis H37Rv. CFU were determined at days 30 and 60 post-infection for the CD4 −/− and CD8 −/− mice and day 30 for the TNF-α KO mice (A) . C57BL/6 mice vaccinated with 5 × 10 4 CFU BCG Pasteur 7 days before infection and the CFU determined 30 days after infection in lungs and spleens (B) . The concentration of and TNF-α, IFN-γ, and IL-2 as determined by cytometric bead array (CBA) at day 30 post-infection when mice were vaccinated 7 and 14 days infection (C) . IFNγ producing cells present in spleen as determined by ELISpot after stimulation with H37Rv Culture filtrate protein (CFP) after infection when mice are vaccinated 7 and 14 days before infection (D) . Experiments were performed with N = 4–5 mice per group and are representative of multiple iterations. * p ≤ 0.05 ** p ≤ 0.01, *** p ≤ 0.001, n.s., not significant.

    Techniques Used: Knock-Out, Mouse Assay, Infection, Concentration Assay, Crocin Bleaching Assay, Enzyme-linked Immunospot

    19) Product Images from "A Neurotrophic Mechanism Directs Sensory Nerve Transit in Cranial Bone"

    Article Title: A Neurotrophic Mechanism Directs Sensory Nerve Transit in Cranial Bone

    Journal: Cell reports

    doi: 10.1016/j.celrep.2020.107696

    Inflammatory Cytokines Induce Ngf Expression within Calvarial Osteoblasts (A and B) Immunohistochemistry for IL-1β, appearing red, on the calvarial injury site within NGF-eGFP reporter sections at (A) d3 and (B) d7 post-injury. Green arrowheads indicate bone-lining eGFP reporter activity, while red arrowheads indicate immunoreactivity within the defect site. White dashed lines indicate bone edges. Inset demonstrates immunoreactivity among uninjured calvarial bone. White scale bar, 50 μm; purple scale bar, 20 μm. (C and D) Immunohistochemistry for TNF-α, appearing red, on the calvarial injury site within NGF-eGFP reporter sections at (C) d3 and (D) d7 post-injury. Green arrowheads indicate bone-lining eGFP reporter activity, while red arrowheads indicate immunoreactivity within the defect site. White dashed lines indicate bone edges. Inset demonstrates immunoreactivity among uninjured calvarial bone. White scale bar, 50 μm; purple scale bar, 20 μm. (E) Ngf expression among calvarial osteoblasts 48 h after treatment with recombinant IL-1β or TNF-α, assessed using qRT-PCR. (F) eGFP expression among NGF-eGFP-derived calvarial osteoblasts 48 h after treatment with IL-1β (1 ng/mL) or TNF-α (25 ng/mL), assessed using qRT-PCR. (G) NF-κB signaling among calvarial osteoblasts 15–60 min after treatment with IL-1β (1 ng/mL) or TNF-α (25 ng/mL), assessed using western blot. All experiments were performed in triplicate. Representative histologic images are from n = 3 mice per time point. †p
    Figure Legend Snippet: Inflammatory Cytokines Induce Ngf Expression within Calvarial Osteoblasts (A and B) Immunohistochemistry for IL-1β, appearing red, on the calvarial injury site within NGF-eGFP reporter sections at (A) d3 and (B) d7 post-injury. Green arrowheads indicate bone-lining eGFP reporter activity, while red arrowheads indicate immunoreactivity within the defect site. White dashed lines indicate bone edges. Inset demonstrates immunoreactivity among uninjured calvarial bone. White scale bar, 50 μm; purple scale bar, 20 μm. (C and D) Immunohistochemistry for TNF-α, appearing red, on the calvarial injury site within NGF-eGFP reporter sections at (C) d3 and (D) d7 post-injury. Green arrowheads indicate bone-lining eGFP reporter activity, while red arrowheads indicate immunoreactivity within the defect site. White dashed lines indicate bone edges. Inset demonstrates immunoreactivity among uninjured calvarial bone. White scale bar, 50 μm; purple scale bar, 20 μm. (E) Ngf expression among calvarial osteoblasts 48 h after treatment with recombinant IL-1β or TNF-α, assessed using qRT-PCR. (F) eGFP expression among NGF-eGFP-derived calvarial osteoblasts 48 h after treatment with IL-1β (1 ng/mL) or TNF-α (25 ng/mL), assessed using qRT-PCR. (G) NF-κB signaling among calvarial osteoblasts 15–60 min after treatment with IL-1β (1 ng/mL) or TNF-α (25 ng/mL), assessed using western blot. All experiments were performed in triplicate. Representative histologic images are from n = 3 mice per time point. †p

    Techniques Used: Expressing, Immunohistochemistry, Activity Assay, Recombinant, Quantitative RT-PCR, Derivative Assay, Western Blot, Mouse Assay

    20) Product Images from "Inhibition of A20 expression in tumor microenvironment exerts anti-tumor effect through inducing myeloid-derived suppressor cells apoptosis"

    Article Title: Inhibition of A20 expression in tumor microenvironment exerts anti-tumor effect through inducing myeloid-derived suppressor cells apoptosis

    Journal: Scientific Reports

    doi: 10.1038/srep16437

    Si-A20 treatment induces the apoptosis of MDSCs in vivo . ( a , b ) Immunohistochemical and ELISA analysis for the expression of TNF-α in tumor in mice treated with PBS, si-SCR and si-A20 (n = 3). Scale bar 100 μm. ( c ) The expression of cleaved caspase-3 in tumor sections in si-A20 treated mice. Tissues were stained with DAPI (blue), antibodies to Gr1 (Alexa-595) and cleaved caspase-3 (FITC) and analyzed by confocal microscopy, Two independent experiments were performed ( n = 5). Scale bar 50 μm. Data were representative of two independent experiments. Data represent means ± SD. * p
    Figure Legend Snippet: Si-A20 treatment induces the apoptosis of MDSCs in vivo . ( a , b ) Immunohistochemical and ELISA analysis for the expression of TNF-α in tumor in mice treated with PBS, si-SCR and si-A20 (n = 3). Scale bar 100 μm. ( c ) The expression of cleaved caspase-3 in tumor sections in si-A20 treated mice. Tissues were stained with DAPI (blue), antibodies to Gr1 (Alexa-595) and cleaved caspase-3 (FITC) and analyzed by confocal microscopy, Two independent experiments were performed ( n = 5). Scale bar 50 μm. Data were representative of two independent experiments. Data represent means ± SD. * p

    Techniques Used: In Vivo, Immunohistochemistry, Enzyme-linked Immunosorbent Assay, Expressing, Mouse Assay, Staining, Confocal Microscopy

    Knockdown of A20 induces the apoptosis of MDSCs through JNK pathway. ( a ) Isolation of MDSCs from tumor tissue was confirmed by flow cytometry. Total number of 30000 cells was analyzed. Numbers illustrated indicate the percentage of the cells in total cells (n = 5). ( b ) mRNA levels of A20 from the isolated MDSCs from spleen and E.G7 tumor were measured (n = 3). ( c ) Fluorescence immunostaining of Gr1 and A20 positive cells in MDSCs isolated from tumor tissue. Scale bar 50 μm. ( d ) Si-A20 treatment induced the apoptosis of MDSC in vitro . Gr1 + CD11b + cells were isolated from tumors in E.G7 tumor-bearing mice and transfected with si-RNA. Apoptosis of MDSCs were confirmed by flow cytometry. Total number of 30000 cells was analyzed. Numbers illustrated indicate the percentage of the cells in total cells. MDSCs were cultured with a mixed medium (TCCM: complete RPMI 1640 = 1:1) with GM-CSF (10 ng/ml). Percentages of the cells in the regions were illustrated. ( e ) Statistic analysis of ( d ). ( f ) Si-A20 induced the apoptosis of MDSCs through JNK pathway. p-JNK, activated caspase-3 and activated caspase-8 were analyzed by western blotting after treatment of MDSCs with si-A20 and TNF-α. Data were representative of two independent experiments. Data represent means ± SD. * p
    Figure Legend Snippet: Knockdown of A20 induces the apoptosis of MDSCs through JNK pathway. ( a ) Isolation of MDSCs from tumor tissue was confirmed by flow cytometry. Total number of 30000 cells was analyzed. Numbers illustrated indicate the percentage of the cells in total cells (n = 5). ( b ) mRNA levels of A20 from the isolated MDSCs from spleen and E.G7 tumor were measured (n = 3). ( c ) Fluorescence immunostaining of Gr1 and A20 positive cells in MDSCs isolated from tumor tissue. Scale bar 50 μm. ( d ) Si-A20 treatment induced the apoptosis of MDSC in vitro . Gr1 + CD11b + cells were isolated from tumors in E.G7 tumor-bearing mice and transfected with si-RNA. Apoptosis of MDSCs were confirmed by flow cytometry. Total number of 30000 cells was analyzed. Numbers illustrated indicate the percentage of the cells in total cells. MDSCs were cultured with a mixed medium (TCCM: complete RPMI 1640 = 1:1) with GM-CSF (10 ng/ml). Percentages of the cells in the regions were illustrated. ( e ) Statistic analysis of ( d ). ( f ) Si-A20 induced the apoptosis of MDSCs through JNK pathway. p-JNK, activated caspase-3 and activated caspase-8 were analyzed by western blotting after treatment of MDSCs with si-A20 and TNF-α. Data were representative of two independent experiments. Data represent means ± SD. * p

    Techniques Used: Isolation, Flow Cytometry, Cytometry, Fluorescence, Immunostaining, In Vitro, Mouse Assay, Transfection, Cell Culture, Western Blot

    21) Product Images from "Inhibitory Effects of Betulinic Acid on LPS-Induced Neuroinflammation Involve M2 Microglial Polarization via CaMKKβ-Dependent AMPK Activation"

    Article Title: Inhibitory Effects of Betulinic Acid on LPS-Induced Neuroinflammation Involve M2 Microglial Polarization via CaMKKβ-Dependent AMPK Activation

    Journal: Frontiers in Molecular Neuroscience

    doi: 10.3389/fnmol.2018.00098

    Knockdown of CaMKKβ by siRNA attenuated BA-promoting effects on M2 polarization of BV-2 microglial cells. Cells were first transfected with 45 nM CaMKKβ siRNA for 24 h, and then treated with BA for 1 h, followed by exposure to LPS stimulation for 6 or 24 h. (A) Transfection for 24 h was able to decrease the expression of CaMKKβ protein. (B,C) CaMKKβ knockdown decreased BA-mediated inhibition of TNF-α production, and mRNA expression of TNF-α and iNOS in LPS-stimulated BV-2 cells. (D–F) CaMKKβ knockdown abolished BA-enhanced IL-10 release and mRNA expression CD206 and Arg-1. Data are presented as means ± SEM of three independent experiments in triplicate. Control group was that treated with control siRNA but not BA or LPS. Two columns sharing the same letter are significantly different ( P
    Figure Legend Snippet: Knockdown of CaMKKβ by siRNA attenuated BA-promoting effects on M2 polarization of BV-2 microglial cells. Cells were first transfected with 45 nM CaMKKβ siRNA for 24 h, and then treated with BA for 1 h, followed by exposure to LPS stimulation for 6 or 24 h. (A) Transfection for 24 h was able to decrease the expression of CaMKKβ protein. (B,C) CaMKKβ knockdown decreased BA-mediated inhibition of TNF-α production, and mRNA expression of TNF-α and iNOS in LPS-stimulated BV-2 cells. (D–F) CaMKKβ knockdown abolished BA-enhanced IL-10 release and mRNA expression CD206 and Arg-1. Data are presented as means ± SEM of three independent experiments in triplicate. Control group was that treated with control siRNA but not BA or LPS. Two columns sharing the same letter are significantly different ( P

    Techniques Used: Transfection, Expressing, Inhibition

    Effects of BA on CaMKKβ/AMPK activation and microglia M2 polarization in the LPS-induced mouse model. The animals were treated with or without BA (30 mg/kg, i.p., once per day) for 4 days, and then injected intraperitoneally with LPS (1 mg/kg) 1 h after the last BA treatment; 6 h after LPS stimulation, animals were sacrificed and brains were then obtained and further isolated for assay. (A) BA significantly increased the phosphorylation of CaMKKβ and AMPK in the cerebral cortex of mouse brain. (B) BA obviously inhibited iNOS expression, but enhanced M2 marker protein CD206 expression. (C,D) BA significantly inhibited the mRNA expression of TNF-α and iNOS, whereas it increased mRNA expression of CD206 and Arg-1. (E,F) Double staining of Iba1 (green) with CD16/32 (red) or CD206 (red) in slices section of cerebral cortex. Nuclei are counterstained with DAPI (blue). Scale bar, 20 μm. Arrows indicate obvious CD16/32 or CD206 positive microglia. Representative images were obtained from one set of experiments, and the three experiments were performed independently. (G,H) The quantity of the percentage of CD16/32 + to Iba1 + cells and CD206 to Iba1 + cells. Data are presented as means ± SEM. Control group was untreated cells. ∗ P
    Figure Legend Snippet: Effects of BA on CaMKKβ/AMPK activation and microglia M2 polarization in the LPS-induced mouse model. The animals were treated with or without BA (30 mg/kg, i.p., once per day) for 4 days, and then injected intraperitoneally with LPS (1 mg/kg) 1 h after the last BA treatment; 6 h after LPS stimulation, animals were sacrificed and brains were then obtained and further isolated for assay. (A) BA significantly increased the phosphorylation of CaMKKβ and AMPK in the cerebral cortex of mouse brain. (B) BA obviously inhibited iNOS expression, but enhanced M2 marker protein CD206 expression. (C,D) BA significantly inhibited the mRNA expression of TNF-α and iNOS, whereas it increased mRNA expression of CD206 and Arg-1. (E,F) Double staining of Iba1 (green) with CD16/32 (red) or CD206 (red) in slices section of cerebral cortex. Nuclei are counterstained with DAPI (blue). Scale bar, 20 μm. Arrows indicate obvious CD16/32 or CD206 positive microglia. Representative images were obtained from one set of experiments, and the three experiments were performed independently. (G,H) The quantity of the percentage of CD16/32 + to Iba1 + cells and CD206 to Iba1 + cells. Data are presented as means ± SEM. Control group was untreated cells. ∗ P

    Techniques Used: Activation Assay, Injection, Isolation, Expressing, Marker, Double Staining

    BA prevented LPS-induced M1 microglia activation and promoted microglia polarization to the M2 phenotype in BV-2 microglial cells. Cells were treated with BA for 1 h, followed by LPS stimulation for 6 or 24 h. The cytokine level was determined by ELISA. The mRNA expression level was determined by qPCR assay. (A) BA inhibited LPS-induced TNF-α release into the culture media of BV-2 cells. (B,C) BA inhibited LPS-induced mRNA expression TNF-α, IL-1β, IL-6, iNOS, CD16, and CD68. (D) BA enhanced IL-10 release into culture media. (E,F) BA prevented the LPS-induced downregulation of TGFβ1, IL-10, CD206, Arg-1, and YM1/2expression. (G,H) BA significantly decreased the iNOS expression, whereas increased the CD206 expression in BV-2 cells, evidenced by Western blot assay and immunofluorescence staining. Scale bar, 25 μm. Data are presented as means ± SEM of three independent experiments in triplicate. Control group was untreated cells. # P
    Figure Legend Snippet: BA prevented LPS-induced M1 microglia activation and promoted microglia polarization to the M2 phenotype in BV-2 microglial cells. Cells were treated with BA for 1 h, followed by LPS stimulation for 6 or 24 h. The cytokine level was determined by ELISA. The mRNA expression level was determined by qPCR assay. (A) BA inhibited LPS-induced TNF-α release into the culture media of BV-2 cells. (B,C) BA inhibited LPS-induced mRNA expression TNF-α, IL-1β, IL-6, iNOS, CD16, and CD68. (D) BA enhanced IL-10 release into culture media. (E,F) BA prevented the LPS-induced downregulation of TGFβ1, IL-10, CD206, Arg-1, and YM1/2expression. (G,H) BA significantly decreased the iNOS expression, whereas increased the CD206 expression in BV-2 cells, evidenced by Western blot assay and immunofluorescence staining. Scale bar, 25 μm. Data are presented as means ± SEM of three independent experiments in triplicate. Control group was untreated cells. # P

    Techniques Used: Activation Assay, Enzyme-linked Immunosorbent Assay, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Immunofluorescence, Staining

    Knockdown of AMPK by siRNA attenuated BA-mediated microglia polarization in BV-2 microglial cells. Cells were first transfected with 40 nM AMPKα siRNA for 24 h, and then treated with BA for 1 h, followed by exposure to LPS stimulation for 6 or 24 h. (A) Transfection for 24 h was able to decrease the expression of AMPKα protein. (B,C) AMPKα knockdown decreased BA-mediated inhibition of TNF-α production, and mRNA expression of TNF-α and iNOS in LPS-stimulated BV-2 cells. (D–F) AMPKα knockdown attenuated BA-enhanced IL-10 release and mRNA expression of CD206 and Arg-1. Data are presented as means ± SEM of three independent experiments in triplicate. Control group was that treated with control siRNA but not BA or LPS. Two columns sharing the same letter are significantly different ( P
    Figure Legend Snippet: Knockdown of AMPK by siRNA attenuated BA-mediated microglia polarization in BV-2 microglial cells. Cells were first transfected with 40 nM AMPKα siRNA for 24 h, and then treated with BA for 1 h, followed by exposure to LPS stimulation for 6 or 24 h. (A) Transfection for 24 h was able to decrease the expression of AMPKα protein. (B,C) AMPKα knockdown decreased BA-mediated inhibition of TNF-α production, and mRNA expression of TNF-α and iNOS in LPS-stimulated BV-2 cells. (D–F) AMPKα knockdown attenuated BA-enhanced IL-10 release and mRNA expression of CD206 and Arg-1. Data are presented as means ± SEM of three independent experiments in triplicate. Control group was that treated with control siRNA but not BA or LPS. Two columns sharing the same letter are significantly different ( P

    Techniques Used: Transfection, Expressing, Inhibition

    AMPK activation is involved in BA-mediated microglial M2 microglia polarization in BV-2 microglial cells. BV-2 cells were pretreated with AMPK inhibitor compound C (CC) for 0.5 h, and then with BA for another 1 h, followed by exposure to LPS stimulation for 6 or 24 h. (A) AMPK inhibitor attenuated BA inhibition of LPS-induced TNF-α production. (B,C) The AMPK inhibitor reversed BA inhibition of LPS-induced mRNA expression of TNF-α, IL-1β, IL-6, iNOS, CD16, and CD68. (D) The AMPK inhibitor abolished BA-enhanced IL-10 release into culture media. (E,F) The AMPK inhibitor abolished BA-enhanced mRNA expression of TGFβ1, IL-10, CD206, Arg-1, and YM1/2. Data are presented as means ± SEM of three independent experiments in triplicate. Control group was untreated cells. # P
    Figure Legend Snippet: AMPK activation is involved in BA-mediated microglial M2 microglia polarization in BV-2 microglial cells. BV-2 cells were pretreated with AMPK inhibitor compound C (CC) for 0.5 h, and then with BA for another 1 h, followed by exposure to LPS stimulation for 6 or 24 h. (A) AMPK inhibitor attenuated BA inhibition of LPS-induced TNF-α production. (B,C) The AMPK inhibitor reversed BA inhibition of LPS-induced mRNA expression of TNF-α, IL-1β, IL-6, iNOS, CD16, and CD68. (D) The AMPK inhibitor abolished BA-enhanced IL-10 release into culture media. (E,F) The AMPK inhibitor abolished BA-enhanced mRNA expression of TGFβ1, IL-10, CD206, Arg-1, and YM1/2. Data are presented as means ± SEM of three independent experiments in triplicate. Control group was untreated cells. # P

    Techniques Used: Activation Assay, Inhibition, Expressing

    CaMKKβ-dependent AMPK activation is involved in BA-mediated microglia polarization in BV-2 microglial cells. BV-2 cells were pretreated with CaMKKβ inhibitor STO-609 (STO) for 0.5 h, and then with BA for another 1 h, followed by exposure to LPS stimulation for 6 or 24 h. (A) STO reversed BA inhibition of LPS-induced TNF-α production. (B,C) STO attenuated BA inhibition of LPS-induced mRNA expression of TNF-α, IL-1β, IL-6, iNOS, CD16, and CD68. (D) STO abolished BA-enhanced M2 marker protein IL-10 release into culture media. (E,F) STO abolished BA-enhanced mRNA expression of TGFβ1, IL-10, CD206, Arg-1, and YM1/2. Data are presented as means ± SEM of three independent experiments in triplicate. Control group was untreated cells. # P
    Figure Legend Snippet: CaMKKβ-dependent AMPK activation is involved in BA-mediated microglia polarization in BV-2 microglial cells. BV-2 cells were pretreated with CaMKKβ inhibitor STO-609 (STO) for 0.5 h, and then with BA for another 1 h, followed by exposure to LPS stimulation for 6 or 24 h. (A) STO reversed BA inhibition of LPS-induced TNF-α production. (B,C) STO attenuated BA inhibition of LPS-induced mRNA expression of TNF-α, IL-1β, IL-6, iNOS, CD16, and CD68. (D) STO abolished BA-enhanced M2 marker protein IL-10 release into culture media. (E,F) STO abolished BA-enhanced mRNA expression of TGFβ1, IL-10, CD206, Arg-1, and YM1/2. Data are presented as means ± SEM of three independent experiments in triplicate. Control group was untreated cells. # P

    Techniques Used: Activation Assay, Inhibition, Expressing, Marker

    22) Product Images from "Inducible MicroRNA-223 Down-Regulation Promotes TLR-Triggered IL-6 and IL-1? Production in Macrophages by Targeting STAT3"

    Article Title: Inducible MicroRNA-223 Down-Regulation Promotes TLR-Triggered IL-6 and IL-1? Production in Macrophages by Targeting STAT3

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0042971

    Knockdown of STAT3 inhibits LPS- and poly (I∶C)-induced IL-6 and IL-1β production. (A) RAW264.7 cells were transfected with STAT3 siRNA or control RNA at a final concentration of 30 nM. After 24 h, STAT3 protein expression was detected by immunoblotting and densitometry analysis was shown. The data shown represent three independent experiments. (B–C) STAT3 siRNA transfected RAW 264.7 cells were stimulated with poly (I∶C) (10 µg/ml) or LPS (100 ng/ml) for 6 h, mRNA levels of IL-6 and TNF-α were determined by qPCR. (D–F) The protein levels of the IL-6, TNF-α and IL-1β at the indicated time points were analyzed by ELISA. Data are the mean ± SD (n = 3) of one representative experiment. Similar results were obtained in three independent experiments. *** p
    Figure Legend Snippet: Knockdown of STAT3 inhibits LPS- and poly (I∶C)-induced IL-6 and IL-1β production. (A) RAW264.7 cells were transfected with STAT3 siRNA or control RNA at a final concentration of 30 nM. After 24 h, STAT3 protein expression was detected by immunoblotting and densitometry analysis was shown. The data shown represent three independent experiments. (B–C) STAT3 siRNA transfected RAW 264.7 cells were stimulated with poly (I∶C) (10 µg/ml) or LPS (100 ng/ml) for 6 h, mRNA levels of IL-6 and TNF-α were determined by qPCR. (D–F) The protein levels of the IL-6, TNF-α and IL-1β at the indicated time points were analyzed by ELISA. Data are the mean ± SD (n = 3) of one representative experiment. Similar results were obtained in three independent experiments. *** p

    Techniques Used: Transfection, Concentration Assay, Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

    MiR-223 negatively regulates LPS or poly (I∶C)-triggered IL-6 and IL-1β production in macrophages. Raw264.7 cells were transfected with mimics or inhibitors of miR-223 and their controls at a final concentration of 30 nM. 24 h later, cells were stimulated with or without LPS (100 ng/ml) or poly (I∶C) (10 µg/ml). (A–F) Supernatants were collected after 12 h to measure IL-6, TNF-α, IL-1β by ELISA. Data are the mean ± SD (n = 3) of three independent experiments. *** p
    Figure Legend Snippet: MiR-223 negatively regulates LPS or poly (I∶C)-triggered IL-6 and IL-1β production in macrophages. Raw264.7 cells were transfected with mimics or inhibitors of miR-223 and their controls at a final concentration of 30 nM. 24 h later, cells were stimulated with or without LPS (100 ng/ml) or poly (I∶C) (10 µg/ml). (A–F) Supernatants were collected after 12 h to measure IL-6, TNF-α, IL-1β by ELISA. Data are the mean ± SD (n = 3) of three independent experiments. *** p

    Techniques Used: Transfection, Concentration Assay, Enzyme-linked Immunosorbent Assay

    MiR-223 inhibits IL-6 mRNA expression but not TNF-α, in TLR-triggered macrophages. RAW264.7 cells were transfected with mimics or inhibitors of miR-223 and their controls at a final concentration of 30 nM. 24 h later, cells were stimulated with or without LPS (100 ng/ml) or poly (I∶C) (10 µg/ml). IL-6 (A, C) and TNF-α (B, D) mRNA levels were measured at 3 h and 6 h post-stimulation by qPCR. Data are the mean ± SD (n = 3) of three independent experiments. *** p
    Figure Legend Snippet: MiR-223 inhibits IL-6 mRNA expression but not TNF-α, in TLR-triggered macrophages. RAW264.7 cells were transfected with mimics or inhibitors of miR-223 and their controls at a final concentration of 30 nM. 24 h later, cells were stimulated with or without LPS (100 ng/ml) or poly (I∶C) (10 µg/ml). IL-6 (A, C) and TNF-α (B, D) mRNA levels were measured at 3 h and 6 h post-stimulation by qPCR. Data are the mean ± SD (n = 3) of three independent experiments. *** p

    Techniques Used: Expressing, Transfection, Concentration Assay, Real-time Polymerase Chain Reaction

    Transfection of miR-223 mimics to STAT3-siRNA pretreated cells abolishes its ability to inhibit IL-6 production. (A, B) RAW264.7 cells were first transfected with STAT3 siRNA or ctrl RNAs, 18 h later, cells were secondly transfected with ctrl or miR-223 mimics. After 24 h, cells were stimulated with LPS or poly (I∶C) for 3 h. IL-6 and TNF-α mRNA expression were analyzed by q-PCR. (C–E) Cells were treated as described in (A, B), IL-6, TNF-α and IL-1β production were measured by ELISA after 12 h stimulation with LPS or poly (I∶C). *** p
    Figure Legend Snippet: Transfection of miR-223 mimics to STAT3-siRNA pretreated cells abolishes its ability to inhibit IL-6 production. (A, B) RAW264.7 cells were first transfected with STAT3 siRNA or ctrl RNAs, 18 h later, cells were secondly transfected with ctrl or miR-223 mimics. After 24 h, cells were stimulated with LPS or poly (I∶C) for 3 h. IL-6 and TNF-α mRNA expression were analyzed by q-PCR. (C–E) Cells were treated as described in (A, B), IL-6, TNF-α and IL-1β production were measured by ELISA after 12 h stimulation with LPS or poly (I∶C). *** p

    Techniques Used: Transfection, Expressing, Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

    23) Product Images from "Lysate of Probiotic Lactobacillus casei DN-114 001 Ameliorates Colitis by Strengthening the Gut Barrier Function and Changing the Gut Microenvironment"

    Article Title: Lysate of Probiotic Lactobacillus casei DN-114 001 Ameliorates Colitis by Strengthening the Gut Barrier Function and Changing the Gut Microenvironment

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0027961

    Pretreatment with Lc changes cytokine production in different parts of the gut. After DSS treatment and 24 hours cultivation, the production of cytokines TNF-α, TGF-β, IL-6, IL-10, IFN-γ differs in various parts of the gut as measured by ELISA. *P
    Figure Legend Snippet: Pretreatment with Lc changes cytokine production in different parts of the gut. After DSS treatment and 24 hours cultivation, the production of cytokines TNF-α, TGF-β, IL-6, IL-10, IFN-γ differs in various parts of the gut as measured by ELISA. *P

    Techniques Used: Enzyme-linked Immunosorbent Assay

    Lc exerts anti-inflammatory effect on LPS-activated macrophage cell line RAW 264.7. (A) Lc decreases the production of TNF-α in LPS-activated macrophages while Lp does not. TNF-α production by cells stimulated with 1 mg/l of LPS is set as 100% and data are expressed as means ± standard error of the mean of three independent experiments. *P
    Figure Legend Snippet: Lc exerts anti-inflammatory effect on LPS-activated macrophage cell line RAW 264.7. (A) Lc decreases the production of TNF-α in LPS-activated macrophages while Lp does not. TNF-α production by cells stimulated with 1 mg/l of LPS is set as 100% and data are expressed as means ± standard error of the mean of three independent experiments. *P

    Techniques Used:

    24) Product Images from "Modulation of cytokines and transcription factors (T-Bet and GATA3) in CD4 enriched cervical cells of Chlamydia trachomatis infected fertile and infertile women upon stimulation with chlamydial inclusion membrane proteins B and C"

    Article Title: Modulation of cytokines and transcription factors (T-Bet and GATA3) in CD4 enriched cervical cells of Chlamydia trachomatis infected fertile and infertile women upon stimulation with chlamydial inclusion membrane proteins B and C

    Journal: Reproductive Biology and Endocrinology : RB & E

    doi: 10.1186/1477-7827-7-84

    mRNA expression of IL-1β, IL-4, IL-5, IL-6, IL-10, IL-12, TNF-α, IFN-γ, GM-CSF, T-Bet and GATA3 in CD4 + cervical cells in GI, GII and GIII after in vitro stimulation with IncB, IncC and CT EB . Real-time RT-PCR analysis of mRNA levels was done at 12 hours post infection where, Group I (GI) comprised of healthy women with no CT infection, Group II (GII) comprised of CT-positive fertile women, Group III (GIII) comprised of CT-positive infertile women. * P
    Figure Legend Snippet: mRNA expression of IL-1β, IL-4, IL-5, IL-6, IL-10, IL-12, TNF-α, IFN-γ, GM-CSF, T-Bet and GATA3 in CD4 + cervical cells in GI, GII and GIII after in vitro stimulation with IncB, IncC and CT EB . Real-time RT-PCR analysis of mRNA levels was done at 12 hours post infection where, Group I (GI) comprised of healthy women with no CT infection, Group II (GII) comprised of CT-positive fertile women, Group III (GIII) comprised of CT-positive infertile women. * P

    Techniques Used: Expressing, In Vitro, Quantitative RT-PCR, Infection

    25) Product Images from "Antiaging and Anxiolytic Effects of Combinatory Formulas Based on Four Medicinal Herbs"

    Article Title: Antiaging and Anxiolytic Effects of Combinatory Formulas Based on Four Medicinal Herbs

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    doi: 10.1155/2017/4624069

    Anti-inflammatory and antioxidative effects in brains of d -galactose-induced aging mice. Different groups of mice were treated as described in Figure 1 . The anti-inflammatory effect was evaluated by measurement of the brain levels of TNF- α (a) and IL-6 (b), and antioxidant effect was evaluated by measurement of the brain level of MDA (c). Data are expressed in mean ± SD. ## ( p
    Figure Legend Snippet: Anti-inflammatory and antioxidative effects in brains of d -galactose-induced aging mice. Different groups of mice were treated as described in Figure 1 . The anti-inflammatory effect was evaluated by measurement of the brain levels of TNF- α (a) and IL-6 (b), and antioxidant effect was evaluated by measurement of the brain level of MDA (c). Data are expressed in mean ± SD. ## ( p

    Techniques Used: Mouse Assay, Multiple Displacement Amplification

    26) Product Images from "Distinct contribution of IL-6, TNF-?, IL-1, and IL-10 to T cell-mediated spontaneous autoimmune arthritis in mice"

    Article Title: Distinct contribution of IL-6, TNF-?, IL-1, and IL-10 to T cell-mediated spontaneous autoimmune arthritis in mice

    Journal: Journal of Clinical Investigation

    doi: 10.1172/JCI200421795

    Expression of cytokines at mRNA and protein levels. ( A ) Quantitative RT-PCR for indicated genes was performed with ankle joints from 8- or 24-week-old SKG or BALB/c mice. Bars show the means ± SD. See Methods for the definition of units. ( B ) Concentrations of indicated cytokines, assessed by ELISA, in the joint fluid of 32-week-old SKG and BALB/c mice ( n = 10 each). Bars show the means ± SD. ( C – F ) Immunohistochemical staining of the synovial tissue of a finger joint of a 4-month-old SKG mouse for IL-1β ( C ), TNF-α ( D ), or IL-6 ( E ), with staining control ( F ) (original magnification, ×40). Insets show higher magnifications of a part of synovium.
    Figure Legend Snippet: Expression of cytokines at mRNA and protein levels. ( A ) Quantitative RT-PCR for indicated genes was performed with ankle joints from 8- or 24-week-old SKG or BALB/c mice. Bars show the means ± SD. See Methods for the definition of units. ( B ) Concentrations of indicated cytokines, assessed by ELISA, in the joint fluid of 32-week-old SKG and BALB/c mice ( n = 10 each). Bars show the means ± SD. ( C – F ) Immunohistochemical staining of the synovial tissue of a finger joint of a 4-month-old SKG mouse for IL-1β ( C ), TNF-α ( D ), or IL-6 ( E ), with staining control ( F ) (original magnification, ×40). Insets show higher magnifications of a part of synovium.

    Techniques Used: Expressing, Quantitative RT-PCR, Mouse Assay, Enzyme-linked Immunosorbent Assay, Immunohistochemistry, Staining

    27) Product Images from "Development of a Live Recombinant BCG Expressing Human Immunodeficiency Virus Type 1 (HIV-1) Gag Using a pMyong2 Vector System: Potential Use As a Novel HIV-1 Vaccine"

    Article Title: Development of a Live Recombinant BCG Expressing Human Immunodeficiency Virus Type 1 (HIV-1) Gag Using a pMyong2 Vector System: Potential Use As a Novel HIV-1 Vaccine

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.00643

    In vivo immune responses induced by p24 recombinant Mycobacterium bovis BCG (rBCG) strains. (A) Schematic of the immunization performed for the in vivo immunological assays. With 4 weeks interval, each group (five mice/group) was immunized twice with wild-type BCG, two rBCG strains, and rSmeg strain. Four weeks after final immunization, mice were sacrificed and their spleens and blood samples were collected for immunological analyses. (B) IFN-γ secretion levels following in vitro stimulation of splenocytes from mice vaccinated with the p24 rBCG strains were detected using an ELISPOT analysis. Representative images of ELISPOT membrane in each group are shown below the graph. (−), negative control; (+), positive control. (C) Levels of the IL-2, IFN-γ, TNF-α, IL-6, and IL-10 cytokines following in vitro stimulation with p24 of splenocytes from mice vaccinated with the p24 rBCG strains were detected using enzyme-linked immunosorbent assay analyses. A total of five mice per group was analyzed. Data are representative of two independent experiments. Mean ± SD are shown. * P
    Figure Legend Snippet: In vivo immune responses induced by p24 recombinant Mycobacterium bovis BCG (rBCG) strains. (A) Schematic of the immunization performed for the in vivo immunological assays. With 4 weeks interval, each group (five mice/group) was immunized twice with wild-type BCG, two rBCG strains, and rSmeg strain. Four weeks after final immunization, mice were sacrificed and their spleens and blood samples were collected for immunological analyses. (B) IFN-γ secretion levels following in vitro stimulation of splenocytes from mice vaccinated with the p24 rBCG strains were detected using an ELISPOT analysis. Representative images of ELISPOT membrane in each group are shown below the graph. (−), negative control; (+), positive control. (C) Levels of the IL-2, IFN-γ, TNF-α, IL-6, and IL-10 cytokines following in vitro stimulation with p24 of splenocytes from mice vaccinated with the p24 rBCG strains were detected using enzyme-linked immunosorbent assay analyses. A total of five mice per group was analyzed. Data are representative of two independent experiments. Mean ± SD are shown. * P

    Techniques Used: In Vivo, Recombinant, Mouse Assay, In Vitro, Enzyme-linked Immunospot, Negative Control, Positive Control, Enzyme-linked Immunosorbent Assay

    28) Product Images from "A Flexible Model of HIV-1 Latency Permitting Evaluation of Many Primary CD4 T-Cell Reservoirs"

    Article Title: A Flexible Model of HIV-1 Latency Permitting Evaluation of Many Primary CD4 T-Cell Reservoirs

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0030176

    Kinetics of HIV-1 reactivation. (A) Latently infected cells were generated as described in Figure 1 with NL4-3 Luciferase virus or NL4-3 mCherry:Luc virus. Cells were either cultured in the presence of media alone or stimulated with 200 nM PMA, 200 nM PMA and 1.5 µM ionomycin, 10 µg/ml PHA, 10 µg/ml PHA with 100 units/ml IL-2, 10 ng/ml TNF-α, anti-CD3+anti-CD28 beads (ratio 1∶1), 100 units/ml IL-2, 62.5 ng/ml IL-7, or 12.5 ng/ml IL-15. Cells were harvested after 48 h of stimulation. RLU shown were normalized based on total protein present in the various cell lysates. All stimulations were performed in triplicate with error bars representing +/− SD. Results are representative of experiments performed with cells from four independent donors. (B) Latently infected cells from the same individual donor were stimulated with anti-CD3+anti-CD28 beads (ratio 1∶1), 200 nM PMA with 1.5 µM ionomycin, or 10 µg/ml PHA with 100 units/ml IL-2 and harvested at the indicated times post-stimulation. Results are representative of kinetic experiments performed with cells isolated from three independent donors.
    Figure Legend Snippet: Kinetics of HIV-1 reactivation. (A) Latently infected cells were generated as described in Figure 1 with NL4-3 Luciferase virus or NL4-3 mCherry:Luc virus. Cells were either cultured in the presence of media alone or stimulated with 200 nM PMA, 200 nM PMA and 1.5 µM ionomycin, 10 µg/ml PHA, 10 µg/ml PHA with 100 units/ml IL-2, 10 ng/ml TNF-α, anti-CD3+anti-CD28 beads (ratio 1∶1), 100 units/ml IL-2, 62.5 ng/ml IL-7, or 12.5 ng/ml IL-15. Cells were harvested after 48 h of stimulation. RLU shown were normalized based on total protein present in the various cell lysates. All stimulations were performed in triplicate with error bars representing +/− SD. Results are representative of experiments performed with cells from four independent donors. (B) Latently infected cells from the same individual donor were stimulated with anti-CD3+anti-CD28 beads (ratio 1∶1), 200 nM PMA with 1.5 µM ionomycin, or 10 µg/ml PHA with 100 units/ml IL-2 and harvested at the indicated times post-stimulation. Results are representative of kinetic experiments performed with cells isolated from three independent donors.

    Techniques Used: Infection, Generated, Luciferase, Cell Culture, Isolation

    29) Product Images from "Tolerogenic Plasmacytoid Dendritic Cells Control Paracoccidioides brasiliensis Infection by Inducting Regulatory T Cells in an IDO-Dependent Manner"

    Article Title: Tolerogenic Plasmacytoid Dendritic Cells Control Paracoccidioides brasiliensis Infection by Inducting Regulatory T Cells in an IDO-Dependent Manner

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1006115

    pDC response to P . brasilienis infection The influx of pDCs to the lungs of P . brasiliensis -infected mice (1×10 6 yeasts cells) was determined by flow cytometry at weeks 2 and 8 post-infection. Lungs were removed, leukocytes obtained and the number of pDCs evaluated. The pDCs were characterized as CD11c + B220 + PDCA + cells as indicated in the gate strategy used (A) and the activation measured by the expression of CD40, CD80, C86 and MHC-II molecules on their surface (B-C). The number of pDCs that migrated to the lungs was also determined by flow cytometric analysis (D). The levels of TNF-α and IFN-β were measured by ELISA in pDC supernatants obtained after18 hr of cell culture (E). Data represent the means ± SEM of at least 8 mice and are representative of two independent experiments with equivalent results (* p
    Figure Legend Snippet: pDC response to P . brasilienis infection The influx of pDCs to the lungs of P . brasiliensis -infected mice (1×10 6 yeasts cells) was determined by flow cytometry at weeks 2 and 8 post-infection. Lungs were removed, leukocytes obtained and the number of pDCs evaluated. The pDCs were characterized as CD11c + B220 + PDCA + cells as indicated in the gate strategy used (A) and the activation measured by the expression of CD40, CD80, C86 and MHC-II molecules on their surface (B-C). The number of pDCs that migrated to the lungs was also determined by flow cytometric analysis (D). The levels of TNF-α and IFN-β were measured by ELISA in pDC supernatants obtained after18 hr of cell culture (E). Data represent the means ± SEM of at least 8 mice and are representative of two independent experiments with equivalent results (* p

    Techniques Used: Infection, Mouse Assay, Flow Cytometry, Cytometry, Activation Assay, Expressing, Enzyme-linked Immunosorbent Assay, Cell Culture

    30) Product Images from "Prostaglandin E2 binding peptide screened by phage displaying: a new therapeutic strategy in rheumatoid arthritis"

    Article Title: Prostaglandin E2 binding peptide screened by phage displaying: a new therapeutic strategy in rheumatoid arthritis

    Journal: Lipids in Health and Disease

    doi: 10.1186/1476-511X-10-75

    Concentration of IL-1β and TNF-α in joint. ( ± s, n = 6) . ELISA showed that the level of IL-1β and TNF-α in the ankle steep were reduced by intraperitoneal PBP. *; P
    Figure Legend Snippet: Concentration of IL-1β and TNF-α in joint. ( ± s, n = 6) . ELISA showed that the level of IL-1β and TNF-α in the ankle steep were reduced by intraperitoneal PBP. *; P

    Techniques Used: Concentration Assay, Enzyme-linked Immunosorbent Assay

    31) Product Images from "Chloroquine attenuates LPS-mediated macrophage activation through miR-669n-regulated SENP6 protein translation"

    Article Title: Chloroquine attenuates LPS-mediated macrophage activation through miR-669n-regulated SENP6 protein translation

    Journal: American Journal of Translational Research

    doi:

    MiR-669n-mediated suppression of SENP6 augments LPS-induced macrophage activation. (A, B) RT-qPCR (A) and Western blot (B) for SENP6 in miR-669n-modified RAW264.7 cells. (C) TNF-α, IL-6 and IFN-γ levels in the conditioned media in miR-669n-modified
    Figure Legend Snippet: MiR-669n-mediated suppression of SENP6 augments LPS-induced macrophage activation. (A, B) RT-qPCR (A) and Western blot (B) for SENP6 in miR-669n-modified RAW264.7 cells. (C) TNF-α, IL-6 and IFN-γ levels in the conditioned media in miR-669n-modified

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

    32) Product Images from "Glycyrrhetinic acid alleviates hepatic inflammation injury in viral hepatitis disease via a HMGB1-TLR4 signaling pathway"

    Article Title: Glycyrrhetinic acid alleviates hepatic inflammation injury in viral hepatitis disease via a HMGB1-TLR4 signaling pathway

    Journal: International Immunopharmacology

    doi: 10.1016/j.intimp.2020.106578

    TLR4 signaling plays a key role in MHV - induced hepatic injury. C57BL/6 wild type (WT) and TLR4 knockout (KO) mice were infected as described previously. Two hours later, WT mice were injected with GA (20 mg/kg, i.p.) every other day (total three times). At 5 days post infection (dpi), mice were sacrificed and serum and liver samples were collected. (A) Effects of TLR4 gene deficiency on the survival of MHV-infected mice. (B) Virus titer of liver homogenates from infected mice. (C) Serum ALT levels at 5 dpi. (D) Representative H E staining of liver tissues at 5 dpi (magnification, ×100); (E-G) IP-10, TNF-α, and IL-6 mRNA expression in the liver of infected mice. (H) Levels of HMGB1 released in the serum in MHV-infected TLR4 KO and GA-treated mice at 5 dpi. The data represent the mean ± SEM, * indicates P
    Figure Legend Snippet: TLR4 signaling plays a key role in MHV - induced hepatic injury. C57BL/6 wild type (WT) and TLR4 knockout (KO) mice were infected as described previously. Two hours later, WT mice were injected with GA (20 mg/kg, i.p.) every other day (total three times). At 5 days post infection (dpi), mice were sacrificed and serum and liver samples were collected. (A) Effects of TLR4 gene deficiency on the survival of MHV-infected mice. (B) Virus titer of liver homogenates from infected mice. (C) Serum ALT levels at 5 dpi. (D) Representative H E staining of liver tissues at 5 dpi (magnification, ×100); (E-G) IP-10, TNF-α, and IL-6 mRNA expression in the liver of infected mice. (H) Levels of HMGB1 released in the serum in MHV-infected TLR4 KO and GA-treated mice at 5 dpi. The data represent the mean ± SEM, * indicates P

    Techniques Used: Knock-Out, Mouse Assay, Infection, Injection, Staining, Expressing

    Neutralizing HMGB1 antibody effectively alleviates liver injury induced by MHV-A59 infection. C57BL/6 mice were infected i.p. with 8 × 10 5 pfu/mouse for survival studies and 1 × 10 4 pfu/mouse for acute infections. Two hours after infection, the animals were administered a HMGB1 neutralizing antibody (2.5 mg/kg, i.p.) every other day (total three times). (A) Serum HMGB1 levels following infection at 3 and 5 dpi. (B) Effects of a HMGB1 neutralizing antibody on the survival of MHV infected mice. (C) Virus titers in liver homogenates of infected mice. (D) Serum ALT levels at 5 dpi; (E) Representative H E staining of liver tissues on 5 dpi (magnification, ×100). (F-H) RNA was isolated from the liver samples levels, and the expression of inflammatory genes IP-10, TNF-α and IL-6 was assessed using qPCR. The data are represented as the mean ± SEM, * indicates P
    Figure Legend Snippet: Neutralizing HMGB1 antibody effectively alleviates liver injury induced by MHV-A59 infection. C57BL/6 mice were infected i.p. with 8 × 10 5 pfu/mouse for survival studies and 1 × 10 4 pfu/mouse for acute infections. Two hours after infection, the animals were administered a HMGB1 neutralizing antibody (2.5 mg/kg, i.p.) every other day (total three times). (A) Serum HMGB1 levels following infection at 3 and 5 dpi. (B) Effects of a HMGB1 neutralizing antibody on the survival of MHV infected mice. (C) Virus titers in liver homogenates of infected mice. (D) Serum ALT levels at 5 dpi; (E) Representative H E staining of liver tissues on 5 dpi (magnification, ×100). (F-H) RNA was isolated from the liver samples levels, and the expression of inflammatory genes IP-10, TNF-α and IL-6 was assessed using qPCR. The data are represented as the mean ± SEM, * indicates P

    Techniques Used: Infection, Mouse Assay, Staining, Isolation, Expressing, Real-time Polymerase Chain Reaction

    Protective effects of GA against MHV infection in mice by suppressing induction of inflammatory cytokines. C57BL/6 mice were infected with 8×10 5 pfu/mouse (i.p.) for survival studies and 1×10 4 pfu/mouse (i.p.) for acute infections. Two hours later, mice were injected with GA (20 mg/kg, i.p.) every other day (total three times). Five days post infection (dpi), mice were sacrificed and serum and liver samples were collected. (A) Effects of GA administration on the survival of MHV-infected mice. (B) Virus titer in liver homogenates. (C) Serum ALT levels at 5 dpi. (D) Representative H E staining of liver tissues at 5 dpi (magnification ×100). (E) RNA was isolated from liver samples at 5 dpi and levels of inflammatory genes IP-10, TNF-α, IL-6 and IL-1β were assessed using qPCR. (F) HMGB1 release assay was performed using the serum from GA treated mice infected by MHV at 5 dpi. (G) Expression of the TLR4 gene in hepatic tissue from infected mice at 5 dpi. The data show fold inductions as compared to the control group (mean ± SEM, * indicates P
    Figure Legend Snippet: Protective effects of GA against MHV infection in mice by suppressing induction of inflammatory cytokines. C57BL/6 mice were infected with 8×10 5 pfu/mouse (i.p.) for survival studies and 1×10 4 pfu/mouse (i.p.) for acute infections. Two hours later, mice were injected with GA (20 mg/kg, i.p.) every other day (total three times). Five days post infection (dpi), mice were sacrificed and serum and liver samples were collected. (A) Effects of GA administration on the survival of MHV-infected mice. (B) Virus titer in liver homogenates. (C) Serum ALT levels at 5 dpi. (D) Representative H E staining of liver tissues at 5 dpi (magnification ×100). (E) RNA was isolated from liver samples at 5 dpi and levels of inflammatory genes IP-10, TNF-α, IL-6 and IL-1β were assessed using qPCR. (F) HMGB1 release assay was performed using the serum from GA treated mice infected by MHV at 5 dpi. (G) Expression of the TLR4 gene in hepatic tissue from infected mice at 5 dpi. The data show fold inductions as compared to the control group (mean ± SEM, * indicates P

    Techniques Used: Infection, Mouse Assay, Injection, Staining, Isolation, Real-time Polymerase Chain Reaction, Release Assay, Expressing

    Licorice in TCM formulas alleviates hepatic injury during long-term oral administration. C57BL/6 female mice were injected with PBS (control) or TCM+licorice solution (Xiaoyao Powder including licorice) or TCM-licorice solution (Xiaoyao Powder excluding licorice) every day (with dose of 1 g/kg, i. g. injection) for 21 days. (A) body weight, (B) serum ALT and (C) serum AST levels on days 5, 10, 15 and 21. (D-E) IP-10 and IL-1β cytokine levels in the serum of different groups on day 21. (F-H) RNA was isolated from liver samples on day 21 and levels of inflammatory genes IP-10, TNF-α and IL-6 were assessed using qPCR. The PCR data represents fold induction as compared to the control group (mean ± SEM, * P
    Figure Legend Snippet: Licorice in TCM formulas alleviates hepatic injury during long-term oral administration. C57BL/6 female mice were injected with PBS (control) or TCM+licorice solution (Xiaoyao Powder including licorice) or TCM-licorice solution (Xiaoyao Powder excluding licorice) every day (with dose of 1 g/kg, i. g. injection) for 21 days. (A) body weight, (B) serum ALT and (C) serum AST levels on days 5, 10, 15 and 21. (D-E) IP-10 and IL-1β cytokine levels in the serum of different groups on day 21. (F-H) RNA was isolated from liver samples on day 21 and levels of inflammatory genes IP-10, TNF-α and IL-6 were assessed using qPCR. The PCR data represents fold induction as compared to the control group (mean ± SEM, * P

    Techniques Used: Mouse Assay, Injection, AST Assay, Isolation, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction

    GA inhibits immune activation triggered by HMGB1 in vitro . (A) HMGB1 release assay in the media of BNL.CL2 hepatocyte cells after MHV infection (MOI =0.5). D2SC cells were co-incubated with HMGB1 (10 μg/ml, HMGB1-Flag protein was purified using anti-Flag M2 Affinity Gel) and different amounts of GA for 6 hours. The culture supernatants were subsequently collected and analyzed for TNF-α (B) and IL-6 (C) production. (D) IP-10 mRNA expression analysis and (E) secreted TNF-α protein levels assay in RAW 264.7 cells stimulated by supernatant from infected BNL.CL2 and treated with GA (100 μg/ml) or anti-HMGB1 Ab (10 μg/ml) for 8 hours. (F) IP-10 secretion analysis in the infected BNL.CL2 cellular supernatants in the presence of GA (100 μg/ml) or HMGB1 neutralizing antibody (10μg/ml). (G) Virus titer of infected BNL.CL2 treated with control, GA (100 μg/ml) or anti-HMGB1 Ab (10 μg/ml). The data are represented as the mean ± SEM. * indicates P
    Figure Legend Snippet: GA inhibits immune activation triggered by HMGB1 in vitro . (A) HMGB1 release assay in the media of BNL.CL2 hepatocyte cells after MHV infection (MOI =0.5). D2SC cells were co-incubated with HMGB1 (10 μg/ml, HMGB1-Flag protein was purified using anti-Flag M2 Affinity Gel) and different amounts of GA for 6 hours. The culture supernatants were subsequently collected and analyzed for TNF-α (B) and IL-6 (C) production. (D) IP-10 mRNA expression analysis and (E) secreted TNF-α protein levels assay in RAW 264.7 cells stimulated by supernatant from infected BNL.CL2 and treated with GA (100 μg/ml) or anti-HMGB1 Ab (10 μg/ml) for 8 hours. (F) IP-10 secretion analysis in the infected BNL.CL2 cellular supernatants in the presence of GA (100 μg/ml) or HMGB1 neutralizing antibody (10μg/ml). (G) Virus titer of infected BNL.CL2 treated with control, GA (100 μg/ml) or anti-HMGB1 Ab (10 μg/ml). The data are represented as the mean ± SEM. * indicates P

    Techniques Used: Activation Assay, In Vitro, Release Assay, Infection, Incubation, Purification, Expressing

    33) Product Images from "The ATP-P2X7 Signaling Axis Is an Essential Sentinel for Intracellular Clostridium difficile Pathogen-Induced Inflammasome Activation"

    Article Title: The ATP-P2X7 Signaling Axis Is an Essential Sentinel for Intracellular Clostridium difficile Pathogen-Induced Inflammasome Activation

    Journal: Frontiers in Cellular and Infection Microbiology

    doi: 10.3389/fcimb.2018.00084

    C. difficile induces caspase-1-dependent IL-1β production. (A,B) Peritoneal macrophages were infected with C. difficile VPI 10463 at increasing MOIs and a time course of infection was evaluated. Mature IL-1β and caspase-1 processing were analyzed by Western blotting. Lower panels showed quantification of mature caspase-1 and IL-1β relative protein level compared with non-infected group. (C) IL-1β and IL-18 production in the supernatant of infected cells were measured by ELISA. (D) Influence of the caspase-1 inhibitor Ac-YVAD-cmk (YVAD, 100 μM) on IL-1β release in infected macrophages by Western blot analysis. (E) The levels of secreted of IL-1β, IL-18, and TNF-α were determined by ELISA after treatment with YVAD. Values represent the mean ± SEM ( N = 3/group). *** p
    Figure Legend Snippet: C. difficile induces caspase-1-dependent IL-1β production. (A,B) Peritoneal macrophages were infected with C. difficile VPI 10463 at increasing MOIs and a time course of infection was evaluated. Mature IL-1β and caspase-1 processing were analyzed by Western blotting. Lower panels showed quantification of mature caspase-1 and IL-1β relative protein level compared with non-infected group. (C) IL-1β and IL-18 production in the supernatant of infected cells were measured by ELISA. (D) Influence of the caspase-1 inhibitor Ac-YVAD-cmk (YVAD, 100 μM) on IL-1β release in infected macrophages by Western blot analysis. (E) The levels of secreted of IL-1β, IL-18, and TNF-α were determined by ELISA after treatment with YVAD. Values represent the mean ± SEM ( N = 3/group). *** p

    Techniques Used: Infection, Western Blot, Enzyme-linked Immunosorbent Assay

    34) Product Images from "Interference of Quercetin on Astragalus Polysaccharide-Induced Macrophage Activation"

    Article Title: Interference of Quercetin on Astragalus Polysaccharide-Induced Macrophage Activation

    Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

    doi: 10.3390/molecules23071563

    Effects of quercetin on nitric oxide (NO) ( A ), TNF-α ( B ) and IL-6 ( C ) production in RAP- and LPS-induced RAW264.7 macrophages. RAW264.7 cells were incubated with quercetin (10, 20 μM) and then treated with RAP (100 μg/mL) or LPS (100 ng/mL) for 24 h. Data from the experiments are expressed as mean ± SD (## p
    Figure Legend Snippet: Effects of quercetin on nitric oxide (NO) ( A ), TNF-α ( B ) and IL-6 ( C ) production in RAP- and LPS-induced RAW264.7 macrophages. RAW264.7 cells were incubated with quercetin (10, 20 μM) and then treated with RAP (100 μg/mL) or LPS (100 ng/mL) for 24 h. Data from the experiments are expressed as mean ± SD (## p

    Techniques Used: Incubation

    Effects of quercetin on inducible nitric oxide synthase (iNOS) ( A ), TNF-α ( B ) and IL-6 ( C ) mRNA expression in RAP-induced RAW264.7 macrophages. RAW264.7 cells were treated with quercetin (10, 20 μM) and then treated with RAP (100 μg/mL). The mRNA expression level was quantified with real time fluorescence PCR. Data from the experiments are expressed as mean ± SD (## p
    Figure Legend Snippet: Effects of quercetin on inducible nitric oxide synthase (iNOS) ( A ), TNF-α ( B ) and IL-6 ( C ) mRNA expression in RAP-induced RAW264.7 macrophages. RAW264.7 cells were treated with quercetin (10, 20 μM) and then treated with RAP (100 μg/mL). The mRNA expression level was quantified with real time fluorescence PCR. Data from the experiments are expressed as mean ± SD (## p

    Techniques Used: Expressing, Fluorescence, Polymerase Chain Reaction

    Effects of quercetin on IL-6 ( A ) and TNF-α ( B ) production in RAP-induced peritoneal macrophages. Peritoneal macrophages cells were incubated with quercetin (10, 20 μM) and then treated with RAP (100 μg/mL) for 24 h. Data from the experiments are expressed as mean ± SD (## p
    Figure Legend Snippet: Effects of quercetin on IL-6 ( A ) and TNF-α ( B ) production in RAP-induced peritoneal macrophages. Peritoneal macrophages cells were incubated with quercetin (10, 20 μM) and then treated with RAP (100 μg/mL) for 24 h. Data from the experiments are expressed as mean ± SD (## p

    Techniques Used: Incubation

    35) Product Images from "Artesunate interacts with Vitamin D receptor to reverse mouse model of sepsis-induced immunosuppression via enhancing autophagy"

    Article Title: Artesunate interacts with Vitamin D receptor to reverse mouse model of sepsis-induced immunosuppression via enhancing autophagy

    Journal: bioRxiv

    doi: 10.1101/2020.02.26.966143

    AS increases pro-inflammatory cytokines release and bacterial clearance within LPS-tolerance macrophages (n = 4). (a) LPS increased the release of TNF-α (a1) and IL-6 (a2) from PMs in a dose-dependent manner. (b) Schematic diagram to establish LPS-tolerance macrophages model. (c) The level of TNF-α (c1) and IL-6 (c2) in LPS-tolerance PMs (n = 5). (d) Effect of AS (10, 20 and 40 μg/mL) treatment on the level of TNF-α (d1, 2) and IL-6 (d3, 4) in LPS-tolerance PMs and RAW264.7 cells (n = 5). (e) Effect of AS treatment (20 μg/mL) on the bacterial load in LPS-tolerance RAW264.7 cells (n = 8). (f) Effect of AS (20 μg/mL) treatment on the level of TNF-α (a1, 3) and IL-6 (a2, 4) in LPS-tolerance THP-1 monocytes and THP-1 derived macrophages (n = 5). (g) Effect of AS treatment (20 μg/mL) on the bacterial load in LPS tolerance THP-1 derived macrophages (n = 8). (* P
    Figure Legend Snippet: AS increases pro-inflammatory cytokines release and bacterial clearance within LPS-tolerance macrophages (n = 4). (a) LPS increased the release of TNF-α (a1) and IL-6 (a2) from PMs in a dose-dependent manner. (b) Schematic diagram to establish LPS-tolerance macrophages model. (c) The level of TNF-α (c1) and IL-6 (c2) in LPS-tolerance PMs (n = 5). (d) Effect of AS (10, 20 and 40 μg/mL) treatment on the level of TNF-α (d1, 2) and IL-6 (d3, 4) in LPS-tolerance PMs and RAW264.7 cells (n = 5). (e) Effect of AS treatment (20 μg/mL) on the bacterial load in LPS-tolerance RAW264.7 cells (n = 8). (f) Effect of AS (20 μg/mL) treatment on the level of TNF-α (a1, 3) and IL-6 (a2, 4) in LPS-tolerance THP-1 monocytes and THP-1 derived macrophages (n = 5). (g) Effect of AS treatment (20 μg/mL) on the bacterial load in LPS tolerance THP-1 derived macrophages (n = 8). (* P

    Techniques Used: Derivative Assay

    Vitamin D receptor is predicted to be a target candidate of AS. (a) A total of 20 underlying signal molecules were selected via the traditional Chinese medicine systems pharmacology database and analysis platform (TCMSP). (b - c) Effect of AS on the relative level of VDR mRNA (b) and protein (c) expression (n = 5). (d) Effect of VDR siRNA (d1) and VDR-KD lentiviral vector (d2) on TNF-α level in LPS tolerance RAW264.7 cells treated with AS (n = 5). (e) Effect of VDR-OE lentiviral vector on TNF-α level in LPS tolerance RAW264.7 cells treated with AS (n = 5). (f1) Schematic diagram of the binding assay designed in our lab. (f2) Effect of V D3 on the binding of AS and VDR tracked by AS-fluorophore (n = 5). AS with fluorophore 12-(7-oxycoumarinyl-ethoxy) dihydro-artemisinin was named AS I, and AS with 12-(-1H-benzo [de] isoquinoline-1, 3(2H)-dione-2-ethoxy) dihydro-artemisinin was named AS II. (g) Effect of V D3 (100 nM) on TNF-α (level in LPS tolerance RAW264.7 cells treated with AS (n = 5). (* P
    Figure Legend Snippet: Vitamin D receptor is predicted to be a target candidate of AS. (a) A total of 20 underlying signal molecules were selected via the traditional Chinese medicine systems pharmacology database and analysis platform (TCMSP). (b - c) Effect of AS on the relative level of VDR mRNA (b) and protein (c) expression (n = 5). (d) Effect of VDR siRNA (d1) and VDR-KD lentiviral vector (d2) on TNF-α level in LPS tolerance RAW264.7 cells treated with AS (n = 5). (e) Effect of VDR-OE lentiviral vector on TNF-α level in LPS tolerance RAW264.7 cells treated with AS (n = 5). (f1) Schematic diagram of the binding assay designed in our lab. (f2) Effect of V D3 on the binding of AS and VDR tracked by AS-fluorophore (n = 5). AS with fluorophore 12-(7-oxycoumarinyl-ethoxy) dihydro-artemisinin was named AS I, and AS with 12-(-1H-benzo [de] isoquinoline-1, 3(2H)-dione-2-ethoxy) dihydro-artemisinin was named AS II. (g) Effect of V D3 (100 nM) on TNF-α (level in LPS tolerance RAW264.7 cells treated with AS (n = 5). (* P

    Techniques Used: Expressing, Plasmid Preparation, Binding Assay

    Knockdown of VDR results in the loss of the effect of AS in vivo . (a) Effect of AS on VDR mRNA level of PMs from CLP mice (n = 5). (b) Effect of AS on VDR protein level of PMs from CLP mice. (c) Change in the effect of AS on the level of TNF-α and IL-6 in the spleen (c1) and lungs (c2) from VDR-KD mice (n = 5). (d) Changes in the level of LC3B-I, LC3B-II, ATG16L, and ATG5 protein expressions in the spleens of VDR-KD mice. (* P
    Figure Legend Snippet: Knockdown of VDR results in the loss of the effect of AS in vivo . (a) Effect of AS on VDR mRNA level of PMs from CLP mice (n = 5). (b) Effect of AS on VDR protein level of PMs from CLP mice. (c) Change in the effect of AS on the level of TNF-α and IL-6 in the spleen (c1) and lungs (c2) from VDR-KD mice (n = 5). (d) Changes in the level of LC3B-I, LC3B-II, ATG16L, and ATG5 protein expressions in the spleens of VDR-KD mice. (* P

    Techniques Used: In Vivo, Mouse Assay

    AS’s effect is autophagy-dependent through VDR in vitro . (a) Effect of ATG16L1 siRNA on TNF-α (a1) and IL-6 (a2) level in LPS tolerance RAW264.7 cells treated with AS (n = 5). (b) Effect of Bafilomycin (Baf) (10 ng/mL) on TNF-α (b1) and IL-6 (b1) level in LPS tolerance RAW264.7 cells treated with AS (n = 5). (c) Effect of 3-MA (5 mM) on TNF-α (c1) and IL-6 (c2) levels in LPS tolerance RAW264.7 cells treated with AS (n = 5). (d) Effect of 3-MA, Ly294002 (10 μM) or Baf on the bacteria clearance in LPS tolerance RAW264.7 cells treated with AS (n = 8). (e1) LPS increased the levels of LC3B-I, LC3B-II, ATG16L1 and ATG5 protein expression in a dose-dependent manner in RAW264.7 cells. (e2) The levels of LC3B-I, LC3B-II, ATG16L1 and ATG5 protein expression over time in RAW264.7 cells treated with LPS (100 ng/mL). The level of expression peaked at 1 h. (f) Representative image of immunofluorescence staining of LC3 in LPS tolerance RAW264.7 cells treated with AS (Bar = 2 μm) (f1) Relative fluorescent puncta indicating LC3 aggregation was quantified from 100 cells, the number in the medium group was normalized as 1 (f2). (g) The expression of LC3B-II, ATG16L1, and ATG5 protein in LPS-tolerance RAW264.7 cells treated with AS. (h) Change of LC3B-II, ATG16L1 and ATG5 protein level in LPS tolerance RAW264.7 cells (VDR-KD) treated with AS. (i) Changes of LC3B-II, ATG16L1 and ATG5 protein levels in LPS tolerance RAW264.7 cells (VDR-OE) treated with AS. (* P
    Figure Legend Snippet: AS’s effect is autophagy-dependent through VDR in vitro . (a) Effect of ATG16L1 siRNA on TNF-α (a1) and IL-6 (a2) level in LPS tolerance RAW264.7 cells treated with AS (n = 5). (b) Effect of Bafilomycin (Baf) (10 ng/mL) on TNF-α (b1) and IL-6 (b1) level in LPS tolerance RAW264.7 cells treated with AS (n = 5). (c) Effect of 3-MA (5 mM) on TNF-α (c1) and IL-6 (c2) levels in LPS tolerance RAW264.7 cells treated with AS (n = 5). (d) Effect of 3-MA, Ly294002 (10 μM) or Baf on the bacteria clearance in LPS tolerance RAW264.7 cells treated with AS (n = 8). (e1) LPS increased the levels of LC3B-I, LC3B-II, ATG16L1 and ATG5 protein expression in a dose-dependent manner in RAW264.7 cells. (e2) The levels of LC3B-I, LC3B-II, ATG16L1 and ATG5 protein expression over time in RAW264.7 cells treated with LPS (100 ng/mL). The level of expression peaked at 1 h. (f) Representative image of immunofluorescence staining of LC3 in LPS tolerance RAW264.7 cells treated with AS (Bar = 2 μm) (f1) Relative fluorescent puncta indicating LC3 aggregation was quantified from 100 cells, the number in the medium group was normalized as 1 (f2). (g) The expression of LC3B-II, ATG16L1, and ATG5 protein in LPS-tolerance RAW264.7 cells treated with AS. (h) Change of LC3B-II, ATG16L1 and ATG5 protein level in LPS tolerance RAW264.7 cells (VDR-KD) treated with AS. (i) Changes of LC3B-II, ATG16L1 and ATG5 protein levels in LPS tolerance RAW264.7 cells (VDR-OE) treated with AS. (* P

    Techniques Used: In Vitro, Expressing, Immunofluorescence, Staining

    AS reverses sepsis-induced immunosuppression in CLP mice. (a) Schematic diagram to establish CLP mice model with secondary bacterial infection. (b) Mortality of CLP mice challenged with PA and treated by AS (10 mg/kg) (n = 20). (c) Effect of AS treatment on the level of TNF-α (c1), IL-6 (c2), and IL-1β (c3) in the serum, spleen, and lungs of CLP mice (n = 8). (d) Effect of AS treatment on the level of TNF-α (d1), IL-6 (d2), and IL-1β (d3) in the serum, spleen, and lungs of CLP mice challenged with PA (n = 8). (e) Effect of AS treatment on the bacterial load in the blood (e1), spleen (e2), and lungs (e3) 4 h after CLP mice were challenged with PA (n = 8). (* P
    Figure Legend Snippet: AS reverses sepsis-induced immunosuppression in CLP mice. (a) Schematic diagram to establish CLP mice model with secondary bacterial infection. (b) Mortality of CLP mice challenged with PA and treated by AS (10 mg/kg) (n = 20). (c) Effect of AS treatment on the level of TNF-α (c1), IL-6 (c2), and IL-1β (c3) in the serum, spleen, and lungs of CLP mice (n = 8). (d) Effect of AS treatment on the level of TNF-α (d1), IL-6 (d2), and IL-1β (d3) in the serum, spleen, and lungs of CLP mice challenged with PA (n = 8). (e) Effect of AS treatment on the bacterial load in the blood (e1), spleen (e2), and lungs (e3) 4 h after CLP mice were challenged with PA (n = 8). (* P

    Techniques Used: Mouse Assay, Infection

    AS inhibits physical interaction between VDR and NF-κB p65 in LPS tolerance macrophages. RAW264.7 cells were treated as described in the legend of figure 2d . (a) The cytoplasm (a1) and nucleus (a2) lysate were used for an IP experiment using anti-VDR antibody, and the associated NF-κB p65 (p65) was detected by immunoblotting (IB). (b) Immunostaining to observe the co-localization of p65 and VDR. P65 was probed using Alexa Fluor 488 (green). VDR was probed using Alexa Fluor 555 (red). Representative images are shown (Bar = 5μm) (b1). The co-localization of VDR and P65 (b2) and karyoplasmic ratio of P65 (b3) was quantified from 100 cells (normalized to medium). (c) P65 level in nucleus lysate were detected using ELISA and IB. (d) Change of P65 level in VDR-KD (d1) or VDR-OE (d2) LPS tolerance RAW264.7 cells treated with AS. (e) Change of TNF-α level in P65-KD (e1) or P65-OE (e2) LPS tolerance RAW264.7 cells treated with AS (n = 5). (* P
    Figure Legend Snippet: AS inhibits physical interaction between VDR and NF-κB p65 in LPS tolerance macrophages. RAW264.7 cells were treated as described in the legend of figure 2d . (a) The cytoplasm (a1) and nucleus (a2) lysate were used for an IP experiment using anti-VDR antibody, and the associated NF-κB p65 (p65) was detected by immunoblotting (IB). (b) Immunostaining to observe the co-localization of p65 and VDR. P65 was probed using Alexa Fluor 488 (green). VDR was probed using Alexa Fluor 555 (red). Representative images are shown (Bar = 5μm) (b1). The co-localization of VDR and P65 (b2) and karyoplasmic ratio of P65 (b3) was quantified from 100 cells (normalized to medium). (c) P65 level in nucleus lysate were detected using ELISA and IB. (d) Change of P65 level in VDR-KD (d1) or VDR-OE (d2) LPS tolerance RAW264.7 cells treated with AS. (e) Change of TNF-α level in P65-KD (e1) or P65-OE (e2) LPS tolerance RAW264.7 cells treated with AS (n = 5). (* P

    Techniques Used: Immunostaining, Enzyme-linked Immunosorbent Assay

    36) Product Images from "Suppression of Tumorigenesis: Modulation of Inflammatory Cytokines by Oral Administration of Microencapsulated Probiotic Yogurt Formulation"

    Article Title: Suppression of Tumorigenesis: Modulation of Inflammatory Cytokines by Oral Administration of Microencapsulated Probiotic Yogurt Formulation

    Journal: International Journal of Inflammation

    doi: 10.4061/2010/894972

    The effect of treatment on luminal cytokine concentration levels found in duodenum, jejunum, and proximal and distal ileum: IL-12 (a), IL-6 (b), TNF- α (c), and IFN- γ (d). Data represent the mean ± SD of concentration levels per group; n = 24. Asterisks: statistical differences ( P
    Figure Legend Snippet: The effect of treatment on luminal cytokine concentration levels found in duodenum, jejunum, and proximal and distal ileum: IL-12 (a), IL-6 (b), TNF- α (c), and IFN- γ (d). Data represent the mean ± SD of concentration levels per group; n = 24. Asterisks: statistical differences ( P

    Techniques Used: Concentration Assay

    37) Product Images from "Platelets protect lung from injury induced by systemic inflammatory response"

    Article Title: Platelets protect lung from injury induced by systemic inflammatory response

    Journal: Scientific Reports

    doi: 10.1038/srep42080

    Platelet transfusion attenuates ECC-induced systemic inflammation. Animals were treated as described in the legend to Fig. 2 , and their plasma was assayed for TNF-α ( A ), TGF-β ( B ), and neutrophil elastase ( D ) using commercial ELISA kits. The activation status of leukocytes was determined as up-regulation of CD11b. For platelet studies, the CD41-positive cell population was analyzed. The percentage of CD11b/CD41 cells, which provides an index of platelet-leukocyte aggregates, was determined by flow cytometry ( C ). Platelet transfusion led to significantly higher TGF-β levels, which were reduced by Tirofiban ( D ). Levels of TGF-β correlated positively with numbers of PLAs ( E ) and negatively with levels of both TNF-α ( F ) and NE ( G ). Data shown are mean ± SEM.
    Figure Legend Snippet: Platelet transfusion attenuates ECC-induced systemic inflammation. Animals were treated as described in the legend to Fig. 2 , and their plasma was assayed for TNF-α ( A ), TGF-β ( B ), and neutrophil elastase ( D ) using commercial ELISA kits. The activation status of leukocytes was determined as up-regulation of CD11b. For platelet studies, the CD41-positive cell population was analyzed. The percentage of CD11b/CD41 cells, which provides an index of platelet-leukocyte aggregates, was determined by flow cytometry ( C ). Platelet transfusion led to significantly higher TGF-β levels, which were reduced by Tirofiban ( D ). Levels of TGF-β correlated positively with numbers of PLAs ( E ) and negatively with levels of both TNF-α ( F ) and NE ( G ). Data shown are mean ± SEM.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Activation Assay, Flow Cytometry, Cytometry

    Platelet transfusion attenuates ECC-induced pulmonary inflammation. Mice were subjected to ECC for 30 min, treated as described in the legend to Fig. 2 , and then the right lung was harvested 1 h later, homogenized, and centrifuged. Samples were assayed for TNF-α ( A ), neutrophil elastase ( B ), and TGF-β ( C ) using commercial ELISA kits. Levels of TGF-β correlated negatively with TNF-α ( D ) and with neutrophil elastase ( E ). Data shown are mean ± SEM.
    Figure Legend Snippet: Platelet transfusion attenuates ECC-induced pulmonary inflammation. Mice were subjected to ECC for 30 min, treated as described in the legend to Fig. 2 , and then the right lung was harvested 1 h later, homogenized, and centrifuged. Samples were assayed for TNF-α ( A ), neutrophil elastase ( B ), and TGF-β ( C ) using commercial ELISA kits. Levels of TGF-β correlated negatively with TNF-α ( D ) and with neutrophil elastase ( E ). Data shown are mean ± SEM.

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

    38) Product Images from "Ilex kaushue and Its Bioactive Component 3,5-Dicaffeoylquinic Acid Protected Mice from Lipopolysaccharide-Induced Acute Lung Injury"

    Article Title: Ilex kaushue and Its Bioactive Component 3,5-Dicaffeoylquinic Acid Protected Mice from Lipopolysaccharide-Induced Acute Lung Injury

    Journal: Scientific Reports

    doi: 10.1038/srep34243

    Content analysis of BALF. Mice were anesthetized and inserted with a plastic cannula into the trachea. After clamping the hilum of left lobe, PBS was injected and recovered to give BALF. Supernatant from centrifuged BALF were applied for total proteins, TNF-α and IL-6 measurement. Cell pellets were resuspended and cytospun for population analysis. ( A ) Examination of cell populations, N: neutrophils, M: macrophages, E: erythrocytes (scale bar = 50 μm in ×200 magnification or 10 μm in ×640 magnification); ( B ) Counts of BALF neutrohpils; ( C ) Levels of total proteins; (D ) Levels of TNF-α; ( E ) Levels of IL-6. Data were presented as mean ± S.E.M. (n = 6). Compared with vehicle group: ## p
    Figure Legend Snippet: Content analysis of BALF. Mice were anesthetized and inserted with a plastic cannula into the trachea. After clamping the hilum of left lobe, PBS was injected and recovered to give BALF. Supernatant from centrifuged BALF were applied for total proteins, TNF-α and IL-6 measurement. Cell pellets were resuspended and cytospun for population analysis. ( A ) Examination of cell populations, N: neutrophils, M: macrophages, E: erythrocytes (scale bar = 50 μm in ×200 magnification or 10 μm in ×640 magnification); ( B ) Counts of BALF neutrohpils; ( C ) Levels of total proteins; (D ) Levels of TNF-α; ( E ) Levels of IL-6. Data were presented as mean ± S.E.M. (n = 6). Compared with vehicle group: ## p

    Techniques Used: Mouse Assay, Injection

    39) Product Images from "P2Y2R-mediated inflammasome activation is involved in tumor progression in breast cancer cells and in radiotherapy-resistant breast cancer"

    Article Title: P2Y2R-mediated inflammasome activation is involved in tumor progression in breast cancer cells and in radiotherapy-resistant breast cancer

    Journal: International Journal of Oncology

    doi: 10.3892/ijo.2018.4552

    MDA-MB-231 cells exhibit increased caspase-1 activity and IL-1β secretion after TNF-α and ATP treatment, which is enhanced in RT-R-MDA-MB-231 cells. (A and B) MDA-MB-231 and RT-R-MDA-MB-231 cells were treated with TNF-α or ATP with the indicated dose and times, and (A) caspase-1 activity and (B) IL-1β secretion levels were then measured as described in the Materials and methods. The values represent the means ± SEM of 3 independent experiments. * P
    Figure Legend Snippet: MDA-MB-231 cells exhibit increased caspase-1 activity and IL-1β secretion after TNF-α and ATP treatment, which is enhanced in RT-R-MDA-MB-231 cells. (A and B) MDA-MB-231 and RT-R-MDA-MB-231 cells were treated with TNF-α or ATP with the indicated dose and times, and (A) caspase-1 activity and (B) IL-1β secretion levels were then measured as described in the Materials and methods. The values represent the means ± SEM of 3 independent experiments. * P

    Techniques Used: Multiple Displacement Amplification, Activity Assay

    MMP-9 activity is modulated by caspase-1 in a P2Y 2 R-dependent manner, in MDA-MB-231 or RT-R-MDA-MB-231 cells. (A) Cells were pre-treated with Ac-YVAD-CMK and then stimulated with TNF-α or ATP for 6 h. MMP-9 gelatinase activity was determined as described in the Materials and methods and quantified. The values represent the means ± SEM of 3 independent experiments. * P
    Figure Legend Snippet: MMP-9 activity is modulated by caspase-1 in a P2Y 2 R-dependent manner, in MDA-MB-231 or RT-R-MDA-MB-231 cells. (A) Cells were pre-treated with Ac-YVAD-CMK and then stimulated with TNF-α or ATP for 6 h. MMP-9 gelatinase activity was determined as described in the Materials and methods and quantified. The values represent the means ± SEM of 3 independent experiments. * P

    Techniques Used: Activity Assay, Multiple Displacement Amplification

    TNF-α and ATP increase the invasive and colony-forming ability of MDA-MB-231 cells, with an enhanced effect in the RT-R-MDA-MB-231 cells, in a caspase-1-dependent manner. (A) Cells were pre-treated with Ac-YVAD-CMK, an irreversible caspase-1 inhibitor and stimulated with TNF-α or ATP for 6 h. Matrigel invasion assay was then performed as described in the Materials and methods. The values represent the means ± SEM of 3 independent experiments. ** P
    Figure Legend Snippet: TNF-α and ATP increase the invasive and colony-forming ability of MDA-MB-231 cells, with an enhanced effect in the RT-R-MDA-MB-231 cells, in a caspase-1-dependent manner. (A) Cells were pre-treated with Ac-YVAD-CMK, an irreversible caspase-1 inhibitor and stimulated with TNF-α or ATP for 6 h. Matrigel invasion assay was then performed as described in the Materials and methods. The values represent the means ± SEM of 3 independent experiments. ** P

    Techniques Used: Multiple Displacement Amplification, Invasion Assay

    Caspase-1 activity or IL-1β secretion induced by TNF-α or ATP is significantly suppressed by P2Y 2 R knockdown or apyrase (an enzyme that rapidly hydrolyzes extracellular nucleotides) in MDA-MB-231 or RT-R-MDA-MB-231 cells. (A) P2Y 2 R mRNA levels were analyzed by RT-PCR to confirm the efficiency of the knockdown in control siRNA (siCTRL)- or P2Y 2 R siRNA (siP2Y 2 R)-transfected cells. (B and C) siCTRL or siP2Y 2 R-transfected cells were stimulated with TNF-α (10 ng/ml) or ATP (10 µ M). (B) Caspase-1 activity and (C) IL-1β secretion were measured 3 h and 24 h after treatment, respectively, as described in the Materials and methods. (D and E) The cells were pre-treated with 10 U/ml apyrase for 1 h and stimulated with TNF-α (10 ng/ml). (D) Caspase-1 activity and (E) IL-1β secretion were measured as described in (A). The values represent the means ± SEM of 3 independent experiments. ** P
    Figure Legend Snippet: Caspase-1 activity or IL-1β secretion induced by TNF-α or ATP is significantly suppressed by P2Y 2 R knockdown or apyrase (an enzyme that rapidly hydrolyzes extracellular nucleotides) in MDA-MB-231 or RT-R-MDA-MB-231 cells. (A) P2Y 2 R mRNA levels were analyzed by RT-PCR to confirm the efficiency of the knockdown in control siRNA (siCTRL)- or P2Y 2 R siRNA (siP2Y 2 R)-transfected cells. (B and C) siCTRL or siP2Y 2 R-transfected cells were stimulated with TNF-α (10 ng/ml) or ATP (10 µ M). (B) Caspase-1 activity and (C) IL-1β secretion were measured 3 h and 24 h after treatment, respectively, as described in the Materials and methods. (D and E) The cells were pre-treated with 10 U/ml apyrase for 1 h and stimulated with TNF-α (10 ng/ml). (D) Caspase-1 activity and (E) IL-1β secretion were measured as described in (A). The values represent the means ± SEM of 3 independent experiments. ** P

    Techniques Used: Activity Assay, Multiple Displacement Amplification, Reverse Transcription Polymerase Chain Reaction, Transfection

    40) Product Images from "VEGF Production by Ly6C+high Monocytes Contributes to Ventilator-Induced Lung Injury"

    Article Title: VEGF Production by Ly6C+high Monocytes Contributes to Ventilator-Induced Lung Injury

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0165317

    Time dependent effects of LPS+HTV on (A) BALF TNF-α, (B) blood TNF-α, (C) BALF IL-6, (D) blood IL-6, (E) BALF VEGF and (F) blood VEGF. Values represent the mean ± SD ( n = 4–6). # p
    Figure Legend Snippet: Time dependent effects of LPS+HTV on (A) BALF TNF-α, (B) blood TNF-α, (C) BALF IL-6, (D) blood IL-6, (E) BALF VEGF and (F) blood VEGF. Values represent the mean ± SD ( n = 4–6). # p

    Techniques Used:

    Related Articles

    SYBR Green Assay:

    Article Title: Mechanical ventilation modulates TLR4 and IRAK-3 in a non-infectious, ventilator-induced lung injury model
    Article Snippet: .. Expression levels of tumour necrosis factor-alpha (TNF -α), interleukin-6 (IL6 ), and IRAK3 genes for all samples were determined by using SYBR green I (Molecular Probes, Leiden, The Netherlands) and the iCycler iQ Real-Time detection System (Bio-Rad Laboratories, CA). .. The β-actin gene was amplified and used as a housekeeping gene.

    Article Title: Xiao-Shen-Formula, a Traditional Chinese Medicine, Improves Glomerular Hyper-Filtration in Diabetic Nephropathy via Inhibiting Arginase Activation and Heparanase Expression
    Article Snippet: .. Gene expression was determined by quantitative PCR with SYBR Green Dye Gene Expression Assays for tumor necrosis factor (TNF-α), vascular cell adhension molecule-1 (VCAM-1), intracellular adhesion molecule 1 (ICALM-1) and Chemokine monocyte chemoattractant protein-1 (MCP-1) and heparanase or TaqMan Gene Expression Assays (for A2, Applied Biosystems), which was performed on a StepOne Plus thermocycler (Applied Biosystems). ..

    Infection:

    Article Title: Mesenchymal stromal cell treatment prevents H9N2 avian influenza virus-induced acute lung injury in mice
    Article Snippet: .. Cytokine measurement Concentrations of the chemokines granulocyte-macrophage colony-stimulating factor (GM-CSF), monocyte chemoattractant protein (MCP-1), keratinocyte chemoattractant (KC), macrophage inflammatory protein-1α (MIP-1α), and monokine induced by IFN-γ (MIG) and the inflammatory cytokines interleukin (IL)-1α, IL-6, IL-10, tumor necrosis factor alpha (TNF-α), and interferon (IFN-γ) in bronchoalveolar lavage fluid (BALF) and serum (five mice from each group) were measured 3 days after H9N2 infection using the Mouse Cytokine Magnetic 20-Plex Panel (Invitrogen) by Luminex 100 (Bio-Rad, USA). ..

    Mouse Assay:

    Article Title: Mesenchymal stromal cell treatment prevents H9N2 avian influenza virus-induced acute lung injury in mice
    Article Snippet: .. Cytokine measurement Concentrations of the chemokines granulocyte-macrophage colony-stimulating factor (GM-CSF), monocyte chemoattractant protein (MCP-1), keratinocyte chemoattractant (KC), macrophage inflammatory protein-1α (MIP-1α), and monokine induced by IFN-γ (MIG) and the inflammatory cytokines interleukin (IL)-1α, IL-6, IL-10, tumor necrosis factor alpha (TNF-α), and interferon (IFN-γ) in bronchoalveolar lavage fluid (BALF) and serum (five mice from each group) were measured 3 days after H9N2 infection using the Mouse Cytokine Magnetic 20-Plex Panel (Invitrogen) by Luminex 100 (Bio-Rad, USA). ..

    Article Title: Skin Allografting Activates Anti-tumor Immunity and Suppresses Growth of Colon Cancer in Mice
    Article Snippet: .. Enzyme-Linked Immunosorbent Assay (ELISA) The serum levels of tumor necrosis factor (TNF)-α and interferon (IFN)-γ in BALB/c mice in each group were measured by the ELISA kit (e Biosciences, San Diego, CA, USA) according to the manufacturer's instructions. .. The optical density (OD) value was measured through the Microplate Reader (Tecan, Männedorf, Switzerland).

    Enzyme-linked Immunosorbent Assay:

    Article Title: Skin Allografting Activates Anti-tumor Immunity and Suppresses Growth of Colon Cancer in Mice
    Article Snippet: .. Enzyme-Linked Immunosorbent Assay (ELISA) The serum levels of tumor necrosis factor (TNF)-α and interferon (IFN)-γ in BALB/c mice in each group were measured by the ELISA kit (e Biosciences, San Diego, CA, USA) according to the manufacturer's instructions. .. The optical density (OD) value was measured through the Microplate Reader (Tecan, Männedorf, Switzerland).

    Article Title: A Zinc-Dependent Metalloproteinase of Brucella abortus Is Required in the Intracellular Adaptation of Macrophages
    Article Snippet: .. TNF-α and IL-6 production was evaluated by ELISA using the commercial kits eBioscience Mouse TNF alpha and Mouse IL-6 ELISA Ready-SET-Go! (Fisher Scientific, MA, United States). .. Final concentrations of cytokines were quantified by standard curves based on the concentration of recombinant mouse TNF-α and IL-6.

    Sandwich ELISA:

    Article Title: The α2 Na+/K+-ATPase isoform mediates LPS-induced neuroinflammation
    Article Snippet: .. The concentrations of TNF-α (#88-7324), IL-1β (#MLB00C), IL-6 (#M6000B), and IFN-γ (#MIF00) were measured by mouse-specific sandwich ELISA according to the manufacturer’s instructions (eBioScience, Santa Clara, California, USA and R & D Systems, Minneapolis, USA) . .. Briefly, samples were added to coated microwells with antibodies against TNF-α, IL-1β, IL-6, and IFN-γ along with a biotin-conjugated antibody (horseradish peroxidases; Mouse IL-1β Conjugate # 893830), polyclonal antibody specific for mouse IL-1β conjugated to horseradish peroxidase with preservatives (R & D Systems), Mouse IL-6 Conjugate # 892665, polyclonal antibody against mouse IL-6 conjugated to horseradish peroxidase with preservatives (R & D Systems), Mouse IFN-γ Conjugate (# 892666, polyclonal antibody specific for mouse IFN-γ conjugated to horseradish peroxidase with preservatives (R & D Systems).

    other:

    Article Title: Dectin-1 Facilitates IL-18 Production for the Generation of Protective Antibodies Against Candida albicans
    Article Snippet: Cytokines MeasurementConcentrations of TNF-α, IL-6, IL-4, IL-13, and IL-18 in spleen and serum homogenates and concentrations of IL-17 and IFN-γ in kidney homogenates were assayed under manufacturer’s instructions by Ready-Set-Go cytokine kits (Invitrogen) in triplicates.

    Expressing:

    Article Title: Mechanical ventilation modulates TLR4 and IRAK-3 in a non-infectious, ventilator-induced lung injury model
    Article Snippet: .. Expression levels of tumour necrosis factor-alpha (TNF -α), interleukin-6 (IL6 ), and IRAK3 genes for all samples were determined by using SYBR green I (Molecular Probes, Leiden, The Netherlands) and the iCycler iQ Real-Time detection System (Bio-Rad Laboratories, CA). .. The β-actin gene was amplified and used as a housekeeping gene.

    Article Title: Inflammation and Extracellular Matrix Degradation Mediated by Activated Transcription Factors Nuclear Factor-?B and Activator Protein-1 in Inflammatory Acne Lesions in Vivo
    Article Snippet: .. mRNA levels of 12 different cytokines [interleukin (IL)-1α, IL-1β, IL-2, IL-4, IL-5, IL-8, IL-10, IL-12 p35, IL12 p40, IL-15, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α] were measured using the TaqMan Cytokine Gene Expression Plate I kit (Applied Biosystems). .. Reverse transcriptase (RT)-PCR was performed with 18s-rRNA as endogenous control, and the data were analyzed using a 7700 sequence detector system (Applied Biosystems).

    Article Title: Xiao-Shen-Formula, a Traditional Chinese Medicine, Improves Glomerular Hyper-Filtration in Diabetic Nephropathy via Inhibiting Arginase Activation and Heparanase Expression
    Article Snippet: .. Gene expression was determined by quantitative PCR with SYBR Green Dye Gene Expression Assays for tumor necrosis factor (TNF-α), vascular cell adhension molecule-1 (VCAM-1), intracellular adhesion molecule 1 (ICALM-1) and Chemokine monocyte chemoattractant protein-1 (MCP-1) and heparanase or TaqMan Gene Expression Assays (for A2, Applied Biosystems), which was performed on a StepOne Plus thermocycler (Applied Biosystems). ..

    Real-time Polymerase Chain Reaction:

    Article Title: Xiao-Shen-Formula, a Traditional Chinese Medicine, Improves Glomerular Hyper-Filtration in Diabetic Nephropathy via Inhibiting Arginase Activation and Heparanase Expression
    Article Snippet: .. Gene expression was determined by quantitative PCR with SYBR Green Dye Gene Expression Assays for tumor necrosis factor (TNF-α), vascular cell adhension molecule-1 (VCAM-1), intracellular adhesion molecule 1 (ICALM-1) and Chemokine monocyte chemoattractant protein-1 (MCP-1) and heparanase or TaqMan Gene Expression Assays (for A2, Applied Biosystems), which was performed on a StepOne Plus thermocycler (Applied Biosystems). ..

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    Thermo Fisher gene exp tnf hs00174128 m1
    Gene expression of pro-inflammatory cytokines ( IL-8 , IL-6 , <t>TNF-α</t> , and IL-1β ) after 15 min, 1 h, and 2 h of CTSS treatment in human corneal epithelial cells (HCE-T cells). The amount of CTSS added corresponded to an activity level found in the 90th–95th percentile of SS patients (18,000 RFU, added to 500 µL of cell medium), as described in detail in Methods . Expression of genes of interest was normalized to expression of the endogenous gene, GAPDH ( n = 3 samples/group, * p ≤ 0.05, data are represented as mean ± SEM and one-way ANOVA with Dunnett’s multiple comparison was used to compare treated to untreated cells).
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    MDSCs compromise macrophage control of E. coli . Bone marrow-derived macrophages were infected with Escherichia coli at an MOI of 20 in the presence or absence of MDSCs (5–13 days of age) at the indicated ratio. Recovery of intracellular bacteria was enumerated at 18 h and shown as mean log 10 recovered colony forming units ± standard error ( a ). Statistical significance in a combined six experiments was determined using a paired samples t test . ( b ) MDSCs were added to cultures where shown at the indicated ratio to macrophages. A combined four experiments are shown. Statistical significance was determined using paired samples t tests . ( c ) Macrophages were cultured as indicated ± MDSCs (ratio of 1:1 with macrophages) in the presence or absence of a soluble receptor to neutralize IL-27 (sIL27R). Five combined experiments are shown. Statistical significance was determined using paired samples t tests . ( d ) The concentration of <t>TNF-α</t> in culture supernatants was determined by ELISA. The presence of bacteria (Ec), sIL-27R, and ratio of MDSCs to macrophages (0.1X or 1X) is indicated. Results from an individual experiment representative of multiple are shown.
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    Effect of valsartan and LCZ696 on renal inflammatory bio-markers including tumor necrosis factor-α <t>(TNF-α)</t> (A), interleukin-1β (IL-1β) (B), interleukin-6 (IL-6) (C) and nuclear factor kappa-B (NF-κB) (D) in STZ-induced diabetic rats ( n = 6 per group). Statistically significant difference: ** p
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    Gene expression of pro-inflammatory cytokines ( IL-8 , IL-6 , TNF-α , and IL-1β ) after 15 min, 1 h, and 2 h of CTSS treatment in human corneal epithelial cells (HCE-T cells). The amount of CTSS added corresponded to an activity level found in the 90th–95th percentile of SS patients (18,000 RFU, added to 500 µL of cell medium), as described in detail in Methods . Expression of genes of interest was normalized to expression of the endogenous gene, GAPDH ( n = 3 samples/group, * p ≤ 0.05, data are represented as mean ± SEM and one-way ANOVA with Dunnett’s multiple comparison was used to compare treated to untreated cells).

    Journal: International Journal of Molecular Sciences

    Article Title: Cathepsin S Alters the Expression of Pro-Inflammatory Cytokines and MMP-9, Partially through Protease—Activated Receptor-2, in Human Corneal Epithelial Cells

    doi: 10.3390/ijms19113530

    Figure Lengend Snippet: Gene expression of pro-inflammatory cytokines ( IL-8 , IL-6 , TNF-α , and IL-1β ) after 15 min, 1 h, and 2 h of CTSS treatment in human corneal epithelial cells (HCE-T cells). The amount of CTSS added corresponded to an activity level found in the 90th–95th percentile of SS patients (18,000 RFU, added to 500 µL of cell medium), as described in detail in Methods . Expression of genes of interest was normalized to expression of the endogenous gene, GAPDH ( n = 3 samples/group, * p ≤ 0.05, data are represented as mean ± SEM and one-way ANOVA with Dunnett’s multiple comparison was used to compare treated to untreated cells).

    Article Snippet: Briefly, samples were preheated at 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. GAPDH (Hs02786624_g1) was run as the internal control and IL-1β (Hs00174097_m1), IL-8 (Hs00174103_m1), IL-6 (Hs00985639_m1), TNF-α (Hs00174128_m1), MMP-9 (Hs00234579_m1), CTSS (Hs00175407_m1), and PAR-2 (Hs00608346_m1) primers were used to measure gene expression.

    Techniques: Expressing, Activity Assay

    CTSS increases IL-1β , IL-8 , IL-6 , and TNF-α gene expression after 2- and 4-hours of treatment in a human corneal epithelial cell line (HCE-T cells) ( A ) IL-1β gene expression without and with CTSS treatment in HCE-T cells; ( B ) IL-8 gene expression without and with CTSS treatment in HCE-T cells; ( C ) IL-6 gene expression without and with CTSS treatment in HCE-T cells; ( D ) TNF-α gene expression without and with CTSS treatment in HCE-T cells. The amount of CTSS added corresponded to an activity level found in the 90th–95th percentile of SS patients (18,000 RFU, added to 500 µL of cell medium), as described in detail in Methods . Expression of genes of interest were normalized to expression of the endogenous gene, GAPDH ( n = 3 samples/group, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, data are represented as mean ± SEM and one-way ANOVA with Dunnett’s multiple comparison was used to compare treated to untreated cells).

    Journal: International Journal of Molecular Sciences

    Article Title: Cathepsin S Alters the Expression of Pro-Inflammatory Cytokines and MMP-9, Partially through Protease—Activated Receptor-2, in Human Corneal Epithelial Cells

    doi: 10.3390/ijms19113530

    Figure Lengend Snippet: CTSS increases IL-1β , IL-8 , IL-6 , and TNF-α gene expression after 2- and 4-hours of treatment in a human corneal epithelial cell line (HCE-T cells) ( A ) IL-1β gene expression without and with CTSS treatment in HCE-T cells; ( B ) IL-8 gene expression without and with CTSS treatment in HCE-T cells; ( C ) IL-6 gene expression without and with CTSS treatment in HCE-T cells; ( D ) TNF-α gene expression without and with CTSS treatment in HCE-T cells. The amount of CTSS added corresponded to an activity level found in the 90th–95th percentile of SS patients (18,000 RFU, added to 500 µL of cell medium), as described in detail in Methods . Expression of genes of interest were normalized to expression of the endogenous gene, GAPDH ( n = 3 samples/group, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, data are represented as mean ± SEM and one-way ANOVA with Dunnett’s multiple comparison was used to compare treated to untreated cells).

    Article Snippet: Briefly, samples were preheated at 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. GAPDH (Hs02786624_g1) was run as the internal control and IL-1β (Hs00174097_m1), IL-8 (Hs00174103_m1), IL-6 (Hs00985639_m1), TNF-α (Hs00174128_m1), MMP-9 (Hs00234579_m1), CTSS (Hs00175407_m1), and PAR-2 (Hs00608346_m1) primers were used to measure gene expression.

    Techniques: Expressing, Activity Assay

    CTSS significantly increases IL-8, IL-6, and TNF-α, IL-1β protein expression in cell culture medium and cell lysates from human corneal epithelial cells (HCE-T cells) at 2, 4, and 8 h of exposure. ( A ) IL-8 protein expression in cell culture medium from HCE-T cells without and with CTSS; ( B ) IL-8 protein expression in cell lysates from HCE-T cells without and with CTSS; ( C ) IL-6 protein expression in cell culture medium from HCE-T cells without and with CTSS; ( D ) IL-6 protein expression in cell lysates from HCE-T cells without and with CTSS; ( E ) TNF-α protein expression in cell culture medium from HCE-T cells without and with CTSS; ( F ) TNF-α protein expression in cell lysates from HCE-T cells without and with CTSS; ( G ) IL-1β protein expression in cell culture medium from HCE-T cells without and with CTSS; ( H ) IL-1β protein expression in cell lysate from HCE-T cells without and with CTSS. The amount of CTSS added corresponded to an activity level found in the 90th–95th percentile of SS patients (18,000 RFU, added to 500 µL of cell medium), as described in detail in Methods . Expression of proteins of interest were normalized to total protein concentration of respective cell culture medium or lysates. a = individual ELISA kit ( n = 3 samples/group, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, data are represented as mean ± SEM and one-way ANOVA with Dunnett’s multiple comparison was used to compare treated to untreated cells).

    Journal: International Journal of Molecular Sciences

    Article Title: Cathepsin S Alters the Expression of Pro-Inflammatory Cytokines and MMP-9, Partially through Protease—Activated Receptor-2, in Human Corneal Epithelial Cells

    doi: 10.3390/ijms19113530

    Figure Lengend Snippet: CTSS significantly increases IL-8, IL-6, and TNF-α, IL-1β protein expression in cell culture medium and cell lysates from human corneal epithelial cells (HCE-T cells) at 2, 4, and 8 h of exposure. ( A ) IL-8 protein expression in cell culture medium from HCE-T cells without and with CTSS; ( B ) IL-8 protein expression in cell lysates from HCE-T cells without and with CTSS; ( C ) IL-6 protein expression in cell culture medium from HCE-T cells without and with CTSS; ( D ) IL-6 protein expression in cell lysates from HCE-T cells without and with CTSS; ( E ) TNF-α protein expression in cell culture medium from HCE-T cells without and with CTSS; ( F ) TNF-α protein expression in cell lysates from HCE-T cells without and with CTSS; ( G ) IL-1β protein expression in cell culture medium from HCE-T cells without and with CTSS; ( H ) IL-1β protein expression in cell lysate from HCE-T cells without and with CTSS. The amount of CTSS added corresponded to an activity level found in the 90th–95th percentile of SS patients (18,000 RFU, added to 500 µL of cell medium), as described in detail in Methods . Expression of proteins of interest were normalized to total protein concentration of respective cell culture medium or lysates. a = individual ELISA kit ( n = 3 samples/group, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, data are represented as mean ± SEM and one-way ANOVA with Dunnett’s multiple comparison was used to compare treated to untreated cells).

    Article Snippet: Briefly, samples were preheated at 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. GAPDH (Hs02786624_g1) was run as the internal control and IL-1β (Hs00174097_m1), IL-8 (Hs00174103_m1), IL-6 (Hs00985639_m1), TNF-α (Hs00174128_m1), MMP-9 (Hs00234579_m1), CTSS (Hs00175407_m1), and PAR-2 (Hs00608346_m1) primers were used to measure gene expression.

    Techniques: Expressing, Cell Culture, Activity Assay, Protein Concentration, Enzyme-linked Immunosorbent Assay

    MDSCs compromise macrophage control of E. coli . Bone marrow-derived macrophages were infected with Escherichia coli at an MOI of 20 in the presence or absence of MDSCs (5–13 days of age) at the indicated ratio. Recovery of intracellular bacteria was enumerated at 18 h and shown as mean log 10 recovered colony forming units ± standard error ( a ). Statistical significance in a combined six experiments was determined using a paired samples t test . ( b ) MDSCs were added to cultures where shown at the indicated ratio to macrophages. A combined four experiments are shown. Statistical significance was determined using paired samples t tests . ( c ) Macrophages were cultured as indicated ± MDSCs (ratio of 1:1 with macrophages) in the presence or absence of a soluble receptor to neutralize IL-27 (sIL27R). Five combined experiments are shown. Statistical significance was determined using paired samples t tests . ( d ) The concentration of TNF-α in culture supernatants was determined by ELISA. The presence of bacteria (Ec), sIL-27R, and ratio of MDSCs to macrophages (0.1X or 1X) is indicated. Results from an individual experiment representative of multiple are shown.

    Journal: Immunology and cell biology

    Article Title: Murine myeloid-derived suppressor cells are a source of elevated levels of interleukin-27 in early life and compromise control of bacterial infection

    doi: 10.1111/imcb.12224

    Figure Lengend Snippet: MDSCs compromise macrophage control of E. coli . Bone marrow-derived macrophages were infected with Escherichia coli at an MOI of 20 in the presence or absence of MDSCs (5–13 days of age) at the indicated ratio. Recovery of intracellular bacteria was enumerated at 18 h and shown as mean log 10 recovered colony forming units ± standard error ( a ). Statistical significance in a combined six experiments was determined using a paired samples t test . ( b ) MDSCs were added to cultures where shown at the indicated ratio to macrophages. A combined four experiments are shown. Statistical significance was determined using paired samples t tests . ( c ) Macrophages were cultured as indicated ± MDSCs (ratio of 1:1 with macrophages) in the presence or absence of a soluble receptor to neutralize IL-27 (sIL27R). Five combined experiments are shown. Statistical significance was determined using paired samples t tests . ( d ) The concentration of TNF-α in culture supernatants was determined by ELISA. The presence of bacteria (Ec), sIL-27R, and ratio of MDSCs to macrophages (0.1X or 1X) is indicated. Results from an individual experiment representative of multiple are shown.

    Article Snippet: IL-27, TNF-α, or IL-6 protein levels in culture supernatants were detected using Ready-SET-Go!™ ELISA kits (ThermoFisher Scientific, Waltham) according to manufacturer protocol.

    Techniques: Derivative Assay, Infection, Cell Culture, Concentration Assay, Enzyme-linked Immunosorbent Assay

    Effect of valsartan and LCZ696 on renal inflammatory bio-markers including tumor necrosis factor-α (TNF-α) (A), interleukin-1β (IL-1β) (B), interleukin-6 (IL-6) (C) and nuclear factor kappa-B (NF-κB) (D) in STZ-induced diabetic rats ( n = 6 per group). Statistically significant difference: ** p

    Journal: PeerJ

    Article Title: LCZ696 mitigates diabetic-induced nephropathy through inhibiting oxidative stress, NF-κB mediated inflammation and glomerulosclerosis in rats

    doi: 10.7717/peerj.9196

    Figure Lengend Snippet: Effect of valsartan and LCZ696 on renal inflammatory bio-markers including tumor necrosis factor-α (TNF-α) (A), interleukin-1β (IL-1β) (B), interleukin-6 (IL-6) (C) and nuclear factor kappa-B (NF-κB) (D) in STZ-induced diabetic rats ( n = 6 per group). Statistically significant difference: ** p

    Article Snippet: Renal levels of IL-1β, TNF-α, IL-6 and NF-κB were determined by following the ELISA techniques (Thermo Scientific, Rockford, IL, USA).

    Techniques:

    Effect of valsartan and LCZ696 on serum pro-inflammatory cytokines including interleukin-6 (IL-6) (A), interleukin-1β (IL-1β) (B), tumor necrosis factor-α (TNF-α) (C) and interleukin-10 (IL-10) (D) in STZ-induced diabetic rats. Data are expressed as the mean ± SEM ( n = 6 per group). Statistically significant difference: ** p

    Journal: PeerJ

    Article Title: LCZ696 mitigates diabetic-induced nephropathy through inhibiting oxidative stress, NF-κB mediated inflammation and glomerulosclerosis in rats

    doi: 10.7717/peerj.9196

    Figure Lengend Snippet: Effect of valsartan and LCZ696 on serum pro-inflammatory cytokines including interleukin-6 (IL-6) (A), interleukin-1β (IL-1β) (B), tumor necrosis factor-α (TNF-α) (C) and interleukin-10 (IL-10) (D) in STZ-induced diabetic rats. Data are expressed as the mean ± SEM ( n = 6 per group). Statistically significant difference: ** p

    Article Snippet: Renal levels of IL-1β, TNF-α, IL-6 and NF-κB were determined by following the ELISA techniques (Thermo Scientific, Rockford, IL, USA).

    Techniques: