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  • 99
    Millipore ultrapure lps
    Ultrapure Lps, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 231 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    InvivoGen ultrapure lps
    Regulation of antibody production by Toll‐like receptor 9 (TLR‐9) in Sle1 mice. A and B , B cell (B220+) activation ( A ) and proliferation ( B ) in splenocytes from young (8–10‐week‐old) Sle1 and Sle1 TLR‐9 −/− mice that were left untreated (media), stimulated with <t>R848</t> (0.01 μg/ml), or stimulated with lipopolysaccharide <t>(LPS;</t> 1 μg/ml). B cell activation was measured by flow cytometry after 24 hours of stimulation and is shown as the percentage of CD69+ cells. B cell proliferation was measured according to the 5,6‐carboxyfluorescein succinimidyl ester dilution after 72 hours of stimulation. C , TLR‐7 expression, measured by intracellular flow cytometry, in mouse splenic B220+ B cells. TLR‐7–deficient Sle1 mice ( Sle1 TLR‐7 −/− ) (n = 4) and fluorescence minus one (FMO) samples were used as negative controls. MFI = median fluorescence intensity. D , Levels of IgG subtypes, measured by Luminex, in Sle1 and Sle1 TLR‐9 −/− mouse culture supernatants collected after 96 hours of incubation. Cultures were left untreated, stimulated with R848, or stimulated with LPS. E , Expression of surface IgG (sIgG) (IgG1/IgG2a/IgG2b/IgG3) on freshly isolated B220+CD19+ splenocytes from Sle1 and Sle1 TLR‐9 −/− mice. F and G , TLR‐7 expression and frequencies of splenic CD138+ plasma/plasmablasts ( F ) and CD11b+ dendritic cells (DCs) ( G ) in Sle1 and Sle1 TLR‐9 −/− mice. In A , B , and D , bars show the mean ± SEM from 2–3 independent experiments (n = 9–15 mice per group). Data were assessed by multiple t ‐tests, and statistical significance was corrected using the Holm‐Sidak method. In C , E , F , and G , data are from 1 representative experiment with 8–10‐week‐old mice (n = 8 Sle1 and 5 Sle1 TLR‐9 −/− mice). Circles represent individual mice; horizontal lines and error bars show the mean ± SEM. Significance was determined by Student's t ‐test. * = P
    Ultrapure Lps, supplied by InvivoGen, used in various techniques. Bioz Stars score: 99/100, based on 2240 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    List Biological Laboratories ultrapure lps
    Bcl-3 promotes survival of DCs. (A) WT and Bcl-3 −/− <t>BMDCs</t> were stimulated with <t>LPS</t> (100ng/ml) for 24h and analyzed with RT 2 profiler apoptosis superarray. Mean of fold-change for n=5 mice/group. (B) WT and Bcl3 −/− BMDCs
    Ultrapure Lps, supplied by List Biological Laboratories, used in various techniques. Bioz Stars score: 92/100, based on 125 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Enzo Biochem ultrapure lps
    Linear assembly of NHE induces K + efflux and activation of the <t>NLRP3</t> inflammasome. a Immunoblot analysis of caspase-1 of WT BMDMs left untreated or <t>LPS-primed</t> and assessed 3 h after treatment with binary combinations of NHE, or after treatment with tripartite NHE. b Release of IL-1β and IL-18, and death of WT BMDMs as treated in a . c , e Immunoblot analysis of caspase-1 of WT BMDMs left untreated or LPS-primed and assessed 3 h after treatment with NHE component added in various orders. d , f Release of IL-1β and IL-18, and death of WT BMDMs as treated in c and e . g Inductively coupled plasma-optical emission spectrometry analysis of intracellular concentrations of K + of BMDMs left untreated or LPS-primed and assessed 2 h after treatment with NHE, or 30 mins after treatment with ATP. h Immunoblot analysis of caspase-1 of WT BMDMs left untreated or LPS-primed and assessed 3 h after treatment with NHE, or 30 mins after treatment with ATP, in the absence (−) or presence (+ ; 50 mM) of extracellular KCl. i Release of IL-1β and IL-18, and death of WT BMDMs as treated in h . NS, not significant, *** P
    Ultrapure Lps, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 92/100, based on 88 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    InvivoGen lipopolysaccharide lps
    Linear assembly of NHE induces K + efflux and activation of the <t>NLRP3</t> inflammasome. a Immunoblot analysis of caspase-1 of WT BMDMs left untreated or <t>LPS-primed</t> and assessed 3 h after treatment with binary combinations of NHE, or after treatment with tripartite NHE. b Release of IL-1β and IL-18, and death of WT BMDMs as treated in a . c , e Immunoblot analysis of caspase-1 of WT BMDMs left untreated or LPS-primed and assessed 3 h after treatment with NHE component added in various orders. d , f Release of IL-1β and IL-18, and death of WT BMDMs as treated in c and e . g Inductively coupled plasma-optical emission spectrometry analysis of intracellular concentrations of K + of BMDMs left untreated or LPS-primed and assessed 2 h after treatment with NHE, or 30 mins after treatment with ATP. h Immunoblot analysis of caspase-1 of WT BMDMs left untreated or LPS-primed and assessed 3 h after treatment with NHE, or 30 mins after treatment with ATP, in the absence (−) or presence (+ ; 50 mM) of extracellular KCl. i Release of IL-1β and IL-18, and death of WT BMDMs as treated in h . NS, not significant, *** P
    Lipopolysaccharide Lps, supplied by InvivoGen, used in various techniques. Bioz Stars score: 99/100, based on 878 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    InvivoGen lps b5 ultrapure
    Antagonistic effects of the activators of TLRs, PI-3K/Akt, ERK1/2, p38/JNK, and NF-κB pathways on the anti-IAV activity of OMT. A549 cells were infected with IAV (MOI = 0.001), treated with or without ribavirin (25 μg/mL) and OMT (50 μg/mL), and simultaneously treated with or without TLR3 activator (polyI:C, 100 ng/mL), TLR4 activator <t>LPS-B5</t> (1 μg/mL), TLR7/8 activator R-848 (10 μg/mL), PI-3K/Akt activator (IGF-1, 100 ng/mL), ERK activator (EGF, 100 ng/mL), p38/JNK activator (Anisomycin, 10 μM), and NF-κB activator (PMA, 1 µg/mL). The test doses of all activators were determined according to previous reports and our preliminary tests. After 48 h, cell viability was determined by the SRB method ( A ) and IAV vRNA levels were determined by a qRT-PCR assay ( B ). Data shown were mean ± SD of three independent experiments performed in triplicate. * p
    Lps B5 Ultrapure, supplied by InvivoGen, used in various techniques. Bioz Stars score: 99/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    InvivoGen ultrapure e coli k12 lps
    Antagonistic effects of the activators of TLRs, PI-3K/Akt, ERK1/2, p38/JNK, and NF-κB pathways on the anti-IAV activity of OMT. A549 cells were infected with IAV (MOI = 0.001), treated with or without ribavirin (25 μg/mL) and OMT (50 μg/mL), and simultaneously treated with or without TLR3 activator (polyI:C, 100 ng/mL), TLR4 activator <t>LPS-B5</t> (1 μg/mL), TLR7/8 activator R-848 (10 μg/mL), PI-3K/Akt activator (IGF-1, 100 ng/mL), ERK activator (EGF, 100 ng/mL), p38/JNK activator (Anisomycin, 10 μM), and NF-κB activator (PMA, 1 µg/mL). The test doses of all activators were determined according to previous reports and our preliminary tests. After 48 h, cell viability was determined by the SRB method ( A ) and IAV vRNA levels were determined by a qRT-PCR assay ( B ). Data shown were mean ± SD of three independent experiments performed in triplicate. * p
    Ultrapure E Coli K12 Lps, supplied by InvivoGen, used in various techniques. Bioz Stars score: 99/100, based on 75 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher ultrapure lps
    Determination of effect of lithium on nitric oxide production on <t>LPS-induced</t> Raw 264.7 cells. Cells were seeded at 6 × 10 6 cell/ml and treated with lithium (5 and 10 mM), 10 mM <t>NaCl,</t> and 100 ng/ml LPS, combining each of the chemicals with LPS for 24 hrs. Cells were stained with DAF2-DA and Hoechst for 20 min at RT. The measurement of the fluorescence was accomplished by using fluorescent inverted Nikon Ti-E microscope at 20x magnification (b). Fluorescence intensity of the pictures was measured using the NIS Element view imaging software (Nikon, Japan) (a). Graphs were executed using GraphPad Prism 4 software and the statistical analysis was carried out using GraphPad InStat software and ANOVA (Tukey–Kramer) ( ∗ p
    Ultrapure Lps, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 97 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    InvivoGen lps rs ultrapure
    Western blot analysis of the levels of <t>TLR4</t> (A, n = 6/group; B, n = 6–8/group), IBA-1 (C, n = 4–7/group; D, n = 7–8/group), and GFAP (E, n = 6–8/group; F, n = 10–11/group) proteins in the rat ipsilateral dorsal lumbar spinal cord (A, C, E) and DRG (B, D, F) after repeated ith. administration of LPS-RSU (20 µg/5 µL, ith. ) on day 7 after chronic constriction injury (CCI). The data are presented as the means ± SEM. Inter-group differences were analyzed using one-way ANOVA followed by Bonferroni’s multiple comparisons test. ∗ p
    Lps Rs Ultrapure, supplied by InvivoGen, used in various techniques. Bioz Stars score: 98/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    InvivoGen reagents ultrapure lps
    Phosphorylation of S5 inhibits NLRP3 activation. (A–C) Quantification of IL-1β and TNF by ELISA in NLRP3-deficient iMOs reconstituted with NLRP3-mCitrine WT or indicated mutations after 3 h <t>LPS</t> priming (for [A] TNF) and stimulated with (B) lethal toxin (6 h) or (C) <t>nigericin</t> (1 h). (A–C) n = 3 ± SEM; (C) *, P
    Reagents Ultrapure Lps, supplied by InvivoGen, used in various techniques. Bioz Stars score: 88/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    InvivoGen ultrapure p gingivalis lps
    Effect of 25(OH)D 3 and 1,25(OH) 2 D 3 on the production of pro-inflammatory mediators by hPdLF with silenced VDR. Cells were transfected with either VDR siRNA or control siRNA and stimulated with P. <t>gingivalis</t> <t>LPS</t> ( Pg LPS, 1 µg/ml) or heat-killed P. gingivalis (hk Pg , 10 8 cells/ml) for 24 h in the presence or in the absence of 25(OH)D 3 or 1,25(OH) 2 D 3 . # means significantly different from control group (2 −▵▵Ct = 1). * means significantly different from cells stimulated with either P. gingivalis LPS or heat-killed P. gingivalis only
    Ultrapure P Gingivalis Lps, supplied by InvivoGen, used in various techniques. Bioz Stars score: 88/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    InvivoGen tlr4 ligand ultrapure lps
    HIV infection increases cell-surface expression of <t>TLR4</t> and CD14 in KCs. (A) Representative FACS analyses of TLR4 expression before (gray histogram) and after (white histogram) HIV infection and <t>LPS</t> treatment. (B) FACS demonstrating quantitative expression level of TLR4 and CD68 in KCs derived from 8 patients directly after isolation and with adherence enrichment and HIV infection KCs. (C) Cumulative data on the TLR4 expression level in CD68-positive cells after HIV infection ( n = 8; P = 0.04). (D) Representative FACS analyses of CD14 expression before (gray histogram) and after (white histogram) HIV infection and LPS treatment. (E) FACS demonstrating quantitative expression level of CD14 and CD68 in KCs derived from 8 patients directly after isolation and with adherence enrichment and HIV infection. (F) Cumulative data on the CD14 expression level in CD68-positive cells after HIV infection ( n = 8; P = 0.0038).
    Tlr4 Ligand Ultrapure Lps, supplied by InvivoGen, used in various techniques. Bioz Stars score: 85/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Regulation of antibody production by Toll‐like receptor 9 (TLR‐9) in Sle1 mice. A and B , B cell (B220+) activation ( A ) and proliferation ( B ) in splenocytes from young (8–10‐week‐old) Sle1 and Sle1 TLR‐9 −/− mice that were left untreated (media), stimulated with R848 (0.01 μg/ml), or stimulated with lipopolysaccharide (LPS; 1 μg/ml). B cell activation was measured by flow cytometry after 24 hours of stimulation and is shown as the percentage of CD69+ cells. B cell proliferation was measured according to the 5,6‐carboxyfluorescein succinimidyl ester dilution after 72 hours of stimulation. C , TLR‐7 expression, measured by intracellular flow cytometry, in mouse splenic B220+ B cells. TLR‐7–deficient Sle1 mice ( Sle1 TLR‐7 −/− ) (n = 4) and fluorescence minus one (FMO) samples were used as negative controls. MFI = median fluorescence intensity. D , Levels of IgG subtypes, measured by Luminex, in Sle1 and Sle1 TLR‐9 −/− mouse culture supernatants collected after 96 hours of incubation. Cultures were left untreated, stimulated with R848, or stimulated with LPS. E , Expression of surface IgG (sIgG) (IgG1/IgG2a/IgG2b/IgG3) on freshly isolated B220+CD19+ splenocytes from Sle1 and Sle1 TLR‐9 −/− mice. F and G , TLR‐7 expression and frequencies of splenic CD138+ plasma/plasmablasts ( F ) and CD11b+ dendritic cells (DCs) ( G ) in Sle1 and Sle1 TLR‐9 −/− mice. In A , B , and D , bars show the mean ± SEM from 2–3 independent experiments (n = 9–15 mice per group). Data were assessed by multiple t ‐tests, and statistical significance was corrected using the Holm‐Sidak method. In C , E , F , and G , data are from 1 representative experiment with 8–10‐week‐old mice (n = 8 Sle1 and 5 Sle1 TLR‐9 −/− mice). Circles represent individual mice; horizontal lines and error bars show the mean ± SEM. Significance was determined by Student's t ‐test. * = P

    Journal: Arthritis & Rheumatology (Hoboken, N.j.)

    Article Title: Toll‐Like Receptor 9 Deficiency Breaks Tolerance to RNA‐Associated Antigens and Up‐Regulates Toll‐Like Receptor 7 Protein in Sle1 Mice

    doi: 10.1002/art.40535

    Figure Lengend Snippet: Regulation of antibody production by Toll‐like receptor 9 (TLR‐9) in Sle1 mice. A and B , B cell (B220+) activation ( A ) and proliferation ( B ) in splenocytes from young (8–10‐week‐old) Sle1 and Sle1 TLR‐9 −/− mice that were left untreated (media), stimulated with R848 (0.01 μg/ml), or stimulated with lipopolysaccharide (LPS; 1 μg/ml). B cell activation was measured by flow cytometry after 24 hours of stimulation and is shown as the percentage of CD69+ cells. B cell proliferation was measured according to the 5,6‐carboxyfluorescein succinimidyl ester dilution after 72 hours of stimulation. C , TLR‐7 expression, measured by intracellular flow cytometry, in mouse splenic B220+ B cells. TLR‐7–deficient Sle1 mice ( Sle1 TLR‐7 −/− ) (n = 4) and fluorescence minus one (FMO) samples were used as negative controls. MFI = median fluorescence intensity. D , Levels of IgG subtypes, measured by Luminex, in Sle1 and Sle1 TLR‐9 −/− mouse culture supernatants collected after 96 hours of incubation. Cultures were left untreated, stimulated with R848, or stimulated with LPS. E , Expression of surface IgG (sIgG) (IgG1/IgG2a/IgG2b/IgG3) on freshly isolated B220+CD19+ splenocytes from Sle1 and Sle1 TLR‐9 −/− mice. F and G , TLR‐7 expression and frequencies of splenic CD138+ plasma/plasmablasts ( F ) and CD11b+ dendritic cells (DCs) ( G ) in Sle1 and Sle1 TLR‐9 −/− mice. In A , B , and D , bars show the mean ± SEM from 2–3 independent experiments (n = 9–15 mice per group). Data were assessed by multiple t ‐tests, and statistical significance was corrected using the Holm‐Sidak method. In C , E , F , and G , data are from 1 representative experiment with 8–10‐week‐old mice (n = 8 Sle1 and 5 Sle1 TLR‐9 −/− mice). Circles represent individual mice; horizontal lines and error bars show the mean ± SEM. Significance was determined by Student's t ‐test. * = P

    Article Snippet: They were plated at 1.5 × 106 cells/ml in round‐bottomed 96‐well plates in the presence of either CpG‐B (ODN 1826), R848, or LPS‐EB Ultrapure (all from InvivoGen) at the indicated concentrations.

    Techniques: Mouse Assay, Activation Assay, Flow Cytometry, Cytometry, Expressing, Fluorescence, Luminex, Incubation, Isolation

    TNF-α production by Lin − progenitor cells differentiated in vitro . Lin − progenitor cells from C57BL/6 mice were cultured in the presence of SCF, FL and IL-7 with medium alone (Medium) or inactivated yeasts of C. albicans ATCC 26555 [120 µg (dry weight) of cells/ml] ( C. albicans ) for 7 days. Afterwards, cells were plated at a density of 250,000 cells in 200 µl of complete cell culture medium, and challenged for 24 h with ultrapure E. coli LPS (0.25 µg/ml), Pam 2 CSK 4 (4 µg/ml), ODN 1585 (1 µg/ml), Curdlan (10 µg/ml) and two amounts of inactivated C. albicans ATCC 26555 yeasts or hyphae: (1) 150 µg (dry weight) of cells/ml or (2) 750 µg (dry weight) of cells/ml. Concentration of TNF-α in cell-free culture supernatants was measured by ELISA. Data represent means ± SD, from triplicates of one representative experiment of three. * P

    Journal: PLoS ONE

    Article Title: Candida albicans Induces Selective Development of Macrophages and Monocyte Derived Dendritic Cells by a TLR2 Dependent Signalling

    doi: 10.1371/journal.pone.0024761

    Figure Lengend Snippet: TNF-α production by Lin − progenitor cells differentiated in vitro . Lin − progenitor cells from C57BL/6 mice were cultured in the presence of SCF, FL and IL-7 with medium alone (Medium) or inactivated yeasts of C. albicans ATCC 26555 [120 µg (dry weight) of cells/ml] ( C. albicans ) for 7 days. Afterwards, cells were plated at a density of 250,000 cells in 200 µl of complete cell culture medium, and challenged for 24 h with ultrapure E. coli LPS (0.25 µg/ml), Pam 2 CSK 4 (4 µg/ml), ODN 1585 (1 µg/ml), Curdlan (10 µg/ml) and two amounts of inactivated C. albicans ATCC 26555 yeasts or hyphae: (1) 150 µg (dry weight) of cells/ml or (2) 750 µg (dry weight) of cells/ml. Concentration of TNF-α in cell-free culture supernatants was measured by ELISA. Data represent means ± SD, from triplicates of one representative experiment of three. * P

    Article Snippet: Stimuli used and preparation of fungal stimuli The stimuli used were Ultrapure E. coli LPS (Invivogen, San Diego, CA), Pam2 CSK4 (Invivogen), ODN 1585 (Invivogen), Curdlan (Wako Chemicals, Richmond, VA) and two inactivated C. albicans ATCC 26555 forms, yeast and hypha, obtained as previously reported , .

    Techniques: In Vitro, Mouse Assay, Cell Culture, Concentration Assay, Enzyme-linked Immunosorbent Assay

    Western blot showing NLRP3 protein expression in H413 epithelial cells treated with different conditions ( P. gingivalis , P. gingivalis LPS, and ATP plus P. gingivalis or P. gingivalis LPS). The trends of the measured NLRP3 bands corresponded to NLRP3 gene expression. * P

    Journal: BMC Oral Health

    Article Title: The activation of pyrin domain-containing-3 inflammasome depends on lipopolysaccharide from Porphyromonas gingivalis and extracellular adenosine triphosphate in cultured oral epithelial cells

    doi: 10.1186/s12903-015-0115-6

    Figure Lengend Snippet: Western blot showing NLRP3 protein expression in H413 epithelial cells treated with different conditions ( P. gingivalis , P. gingivalis LPS, and ATP plus P. gingivalis or P. gingivalis LPS). The trends of the measured NLRP3 bands corresponded to NLRP3 gene expression. * P

    Article Snippet: Cell treatment Confluent H413 cell cultures (5 × 106 cells in T-25 cm2 flasks) were washed three times with PBS and infected with P. gingivalis at a multiplicity of infection (MOI) of 100 bacterial cells per one epithelial cell [ ] for 2 and 4 h [ , ], or stimulated with 1-μg/mL ultrapure lipopolysaccharide (LPS) from P. gingivalis (Invivogen, San Diego, CA, USA) in the cell growth media for 2 and 4 h. Uninfected and non-stimulated cells served as controls.

    Techniques: Western Blot, Expressing

    a Changes in pro-IL-18 mRNA levels in H413 cells with different treatments of P. gingivalis LPS stimuli and ATP plus P. gingivalis infection or P. gingivalis LPS. b ELISA data showing mature IL-18 protein released from H413 cells after different treatments. * P

    Journal: BMC Oral Health

    Article Title: The activation of pyrin domain-containing-3 inflammasome depends on lipopolysaccharide from Porphyromonas gingivalis and extracellular adenosine triphosphate in cultured oral epithelial cells

    doi: 10.1186/s12903-015-0115-6

    Figure Lengend Snippet: a Changes in pro-IL-18 mRNA levels in H413 cells with different treatments of P. gingivalis LPS stimuli and ATP plus P. gingivalis infection or P. gingivalis LPS. b ELISA data showing mature IL-18 protein released from H413 cells after different treatments. * P

    Article Snippet: Cell treatment Confluent H413 cell cultures (5 × 106 cells in T-25 cm2 flasks) were washed three times with PBS and infected with P. gingivalis at a multiplicity of infection (MOI) of 100 bacterial cells per one epithelial cell [ ] for 2 and 4 h [ , ], or stimulated with 1-μg/mL ultrapure lipopolysaccharide (LPS) from P. gingivalis (Invivogen, San Diego, CA, USA) in the cell growth media for 2 and 4 h. Uninfected and non-stimulated cells served as controls.

    Techniques: Infection, Enzyme-linked Immunosorbent Assay

    Gene expressions of NLRP3、ASC and caspase-1. Significant changes in inflammasome genes encoding NLRP3 ( a ), associated adaptor protein (ASC) ( b ), and caspase-1 ( c ) with different treatments of P. gingivalis infection, P. gingivalis LPS stimuli, and ATP plus P. gingivalis or P. gingivalis LPS in H413 epithelial cells. * P

    Journal: BMC Oral Health

    Article Title: The activation of pyrin domain-containing-3 inflammasome depends on lipopolysaccharide from Porphyromonas gingivalis and extracellular adenosine triphosphate in cultured oral epithelial cells

    doi: 10.1186/s12903-015-0115-6

    Figure Lengend Snippet: Gene expressions of NLRP3、ASC and caspase-1. Significant changes in inflammasome genes encoding NLRP3 ( a ), associated adaptor protein (ASC) ( b ), and caspase-1 ( c ) with different treatments of P. gingivalis infection, P. gingivalis LPS stimuli, and ATP plus P. gingivalis or P. gingivalis LPS in H413 epithelial cells. * P

    Article Snippet: Cell treatment Confluent H413 cell cultures (5 × 106 cells in T-25 cm2 flasks) were washed three times with PBS and infected with P. gingivalis at a multiplicity of infection (MOI) of 100 bacterial cells per one epithelial cell [ ] for 2 and 4 h [ , ], or stimulated with 1-μg/mL ultrapure lipopolysaccharide (LPS) from P. gingivalis (Invivogen, San Diego, CA, USA) in the cell growth media for 2 and 4 h. Uninfected and non-stimulated cells served as controls.

    Techniques: Infection

    Innate immune response to P. gingivalis infection, or P. gingivalis LPS and ATP stimulation in epithelial cells. In the left panel, when cells are stimulated with P. gingivalis infection or P. gingivalis LPS without ATP pretreatment, the NLRP3 inflammasome complex is inhibited until P. gingivalis LPS stimulation, except for caspase-1. IL-1β production is not NLRP3 inflammasome dependent, however IL-18 secretion relies on NLRP3 and ASC activation. In the right panel, cells pretreated with ATP, which activates NLRP3 inflammasome, results in the subsequent release of the cytokines IL-1β and IL-18 in P. gingivalis infected cells. ATP can also promote caspase-1 activation upon P. gingivalis LPS stimulation. Secretion of IL-1β is always consistent with caspase-1 activation after stimulation with P. gingivalis LPS

    Journal: BMC Oral Health

    Article Title: The activation of pyrin domain-containing-3 inflammasome depends on lipopolysaccharide from Porphyromonas gingivalis and extracellular adenosine triphosphate in cultured oral epithelial cells

    doi: 10.1186/s12903-015-0115-6

    Figure Lengend Snippet: Innate immune response to P. gingivalis infection, or P. gingivalis LPS and ATP stimulation in epithelial cells. In the left panel, when cells are stimulated with P. gingivalis infection or P. gingivalis LPS without ATP pretreatment, the NLRP3 inflammasome complex is inhibited until P. gingivalis LPS stimulation, except for caspase-1. IL-1β production is not NLRP3 inflammasome dependent, however IL-18 secretion relies on NLRP3 and ASC activation. In the right panel, cells pretreated with ATP, which activates NLRP3 inflammasome, results in the subsequent release of the cytokines IL-1β and IL-18 in P. gingivalis infected cells. ATP can also promote caspase-1 activation upon P. gingivalis LPS stimulation. Secretion of IL-1β is always consistent with caspase-1 activation after stimulation with P. gingivalis LPS

    Article Snippet: Cell treatment Confluent H413 cell cultures (5 × 106 cells in T-25 cm2 flasks) were washed three times with PBS and infected with P. gingivalis at a multiplicity of infection (MOI) of 100 bacterial cells per one epithelial cell [ ] for 2 and 4 h [ , ], or stimulated with 1-μg/mL ultrapure lipopolysaccharide (LPS) from P. gingivalis (Invivogen, San Diego, CA, USA) in the cell growth media for 2 and 4 h. Uninfected and non-stimulated cells served as controls.

    Techniques: Infection, Activation Assay

    Bcl-3 promotes survival of DCs. (A) WT and Bcl-3 −/− BMDCs were stimulated with LPS (100ng/ml) for 24h and analyzed with RT 2 profiler apoptosis superarray. Mean of fold-change for n=5 mice/group. (B) WT and Bcl3 −/− BMDCs

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: The NF-κB regulator Bcl-3 governs dendritic cell antigen presentation functions in adaptive immunity

    doi: 10.4049/jimmunol.1401505

    Figure Lengend Snippet: Bcl-3 promotes survival of DCs. (A) WT and Bcl-3 −/− BMDCs were stimulated with LPS (100ng/ml) for 24h and analyzed with RT 2 profiler apoptosis superarray. Mean of fold-change for n=5 mice/group. (B) WT and Bcl3 −/− BMDCs

    Article Snippet: BMDCs were stimulated with ultrapure LPS ( Escherichia coli 0111:B4; List Biological Laboratories).

    Techniques: Mouse Assay

    Bcl-3 is required for efficient DC-mediated cross-priming in vitro and in vivo. (A) WT and Bcl3 −/− BMDCs were stimulated with LPS (100ng/ml) o.n., pulsed with OVA (100 μg/ml) for 3h, and co-cultured with CFSE-labeled OT-I T cells

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: The NF-κB regulator Bcl-3 governs dendritic cell antigen presentation functions in adaptive immunity

    doi: 10.4049/jimmunol.1401505

    Figure Lengend Snippet: Bcl-3 is required for efficient DC-mediated cross-priming in vitro and in vivo. (A) WT and Bcl3 −/− BMDCs were stimulated with LPS (100ng/ml) o.n., pulsed with OVA (100 μg/ml) for 3h, and co-cultured with CFSE-labeled OT-I T cells

    Article Snippet: BMDCs were stimulated with ultrapure LPS ( Escherichia coli 0111:B4; List Biological Laboratories).

    Techniques: In Vitro, In Vivo, Cell Culture, Labeling

    Bcl-3 contributes to BMDC maturation but is dispensable for inflammatory cytokine production. (A ) WT and Bcl-3 −/− BMDCs were left unstimulated or stimulated with LPS (100ng/ml). 24h later BMDCs were stained for markers indicated, and analyzed

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: The NF-κB regulator Bcl-3 governs dendritic cell antigen presentation functions in adaptive immunity

    doi: 10.4049/jimmunol.1401505

    Figure Lengend Snippet: Bcl-3 contributes to BMDC maturation but is dispensable for inflammatory cytokine production. (A ) WT and Bcl-3 −/− BMDCs were left unstimulated or stimulated with LPS (100ng/ml). 24h later BMDCs were stained for markers indicated, and analyzed

    Article Snippet: BMDCs were stimulated with ultrapure LPS ( Escherichia coli 0111:B4; List Biological Laboratories).

    Techniques: Staining

    Bcl-3 is required for efficient BMDC-mediated CD4 priming in vitro and in vivo. (A) WT and Bcl3 −/− BMDCs were loaded with different doses of ovalbumin (OVA) and stimulated with LPS (100ng/ml) overnight (o.n.), then co-cultured with CFSE-labeled

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: The NF-κB regulator Bcl-3 governs dendritic cell antigen presentation functions in adaptive immunity

    doi: 10.4049/jimmunol.1401505

    Figure Lengend Snippet: Bcl-3 is required for efficient BMDC-mediated CD4 priming in vitro and in vivo. (A) WT and Bcl3 −/− BMDCs were loaded with different doses of ovalbumin (OVA) and stimulated with LPS (100ng/ml) overnight (o.n.), then co-cultured with CFSE-labeled

    Article Snippet: BMDCs were stimulated with ultrapure LPS ( Escherichia coli 0111:B4; List Biological Laboratories).

    Techniques: In Vitro, In Vivo, Cell Culture, Labeling

    Linear assembly of NHE induces K + efflux and activation of the NLRP3 inflammasome. a Immunoblot analysis of caspase-1 of WT BMDMs left untreated or LPS-primed and assessed 3 h after treatment with binary combinations of NHE, or after treatment with tripartite NHE. b Release of IL-1β and IL-18, and death of WT BMDMs as treated in a . c , e Immunoblot analysis of caspase-1 of WT BMDMs left untreated or LPS-primed and assessed 3 h after treatment with NHE component added in various orders. d , f Release of IL-1β and IL-18, and death of WT BMDMs as treated in c and e . g Inductively coupled plasma-optical emission spectrometry analysis of intracellular concentrations of K + of BMDMs left untreated or LPS-primed and assessed 2 h after treatment with NHE, or 30 mins after treatment with ATP. h Immunoblot analysis of caspase-1 of WT BMDMs left untreated or LPS-primed and assessed 3 h after treatment with NHE, or 30 mins after treatment with ATP, in the absence (−) or presence (+ ; 50 mM) of extracellular KCl. i Release of IL-1β and IL-18, and death of WT BMDMs as treated in h . NS, not significant, *** P

    Journal: Nature Communications

    Article Title: Bacillus cereus non-haemolytic enterotoxin activates the NLRP3 inflammasome

    doi: 10.1038/s41467-020-14534-3

    Figure Lengend Snippet: Linear assembly of NHE induces K + efflux and activation of the NLRP3 inflammasome. a Immunoblot analysis of caspase-1 of WT BMDMs left untreated or LPS-primed and assessed 3 h after treatment with binary combinations of NHE, or after treatment with tripartite NHE. b Release of IL-1β and IL-18, and death of WT BMDMs as treated in a . c , e Immunoblot analysis of caspase-1 of WT BMDMs left untreated or LPS-primed and assessed 3 h after treatment with NHE component added in various orders. d , f Release of IL-1β and IL-18, and death of WT BMDMs as treated in c and e . g Inductively coupled plasma-optical emission spectrometry analysis of intracellular concentrations of K + of BMDMs left untreated or LPS-primed and assessed 2 h after treatment with NHE, or 30 mins after treatment with ATP. h Immunoblot analysis of caspase-1 of WT BMDMs left untreated or LPS-primed and assessed 3 h after treatment with NHE, or 30 mins after treatment with ATP, in the absence (−) or presence (+ ; 50 mM) of extracellular KCl. i Release of IL-1β and IL-18, and death of WT BMDMs as treated in h . NS, not significant, *** P

    Article Snippet: To activate the canonical NLRP3 inflammasome, BMDMs were primed with 500 ng/ml ultrapure LPS from E. coli (ALX-581-014-L002, Enzo Life Sciences) for 4 h and stimulated with 5 mM ATP (10127531001, Roche) for 45 min. To activate the NLRC4 inflammasome, BMDMs were infected with S .

    Techniques: Activation Assay

    The transmembrane domain in NHE-C is essential for inflammasome activation and cell death. a Immunoblot analysis of caspase-1 of WT or Nlrp3 −/− BMDMs left untreated (Med.) or LPS-primed and assessed 3 h after treatment with C + B + A (NHE WT ) or C mut. + B + A (NHE mut. ). b Release of IL-1β and IL-18, and death of WT or Nlrp3 −/− BMDMs as treated in a . c Inductively coupled plasma-optical emission spectrometry analysis of intracellular concentrations of K + of BMDMs left untreated or assessed 3 h after treatment with NHE WT or NHE mut. . d Circular dichroism analysis of the secondary structures of C WT or C mut. . e IncuCyte live-imaging analysis of the viability of WT BMDMs left untreated or LPS-primed and assessed after treatment with NHE WT or NHE mut. . f Scanning electron microscopy analysis of WT BMDMs left untreated or LPS-primed and assessed 3 h after treatment with NHE WT or NHE mut. . Scale bar, 2 μm f . NS, not significant, *** P

    Journal: Nature Communications

    Article Title: Bacillus cereus non-haemolytic enterotoxin activates the NLRP3 inflammasome

    doi: 10.1038/s41467-020-14534-3

    Figure Lengend Snippet: The transmembrane domain in NHE-C is essential for inflammasome activation and cell death. a Immunoblot analysis of caspase-1 of WT or Nlrp3 −/− BMDMs left untreated (Med.) or LPS-primed and assessed 3 h after treatment with C + B + A (NHE WT ) or C mut. + B + A (NHE mut. ). b Release of IL-1β and IL-18, and death of WT or Nlrp3 −/− BMDMs as treated in a . c Inductively coupled plasma-optical emission spectrometry analysis of intracellular concentrations of K + of BMDMs left untreated or assessed 3 h after treatment with NHE WT or NHE mut. . d Circular dichroism analysis of the secondary structures of C WT or C mut. . e IncuCyte live-imaging analysis of the viability of WT BMDMs left untreated or LPS-primed and assessed after treatment with NHE WT or NHE mut. . f Scanning electron microscopy analysis of WT BMDMs left untreated or LPS-primed and assessed 3 h after treatment with NHE WT or NHE mut. . Scale bar, 2 μm f . NS, not significant, *** P

    Article Snippet: To activate the canonical NLRP3 inflammasome, BMDMs were primed with 500 ng/ml ultrapure LPS from E. coli (ALX-581-014-L002, Enzo Life Sciences) for 4 h and stimulated with 5 mM ATP (10127531001, Roche) for 45 min. To activate the NLRC4 inflammasome, BMDMs were infected with S .

    Techniques: Activation Assay, Imaging, Electron Microscopy

    NHE forms pores in the cell membrane. a Schematic of toxin-induced liposomal rupture and leakage of dye. b Colorimetric analysis of liposomes left untreated, sonicated for 5 mins at 100 amplitude (CTRL) or assessed 30 mins after treatment with individual NHE subunits (C, B, or A), heat-inactivated NHE (Heat), NHE, HBL, buffer, or bovine serum albumin (BSA). c The absorbance (OD) of residual dye following treatment as in b . d Immunoblot analysis of caspase-1, gasdermin D, and IL-1β of WT BMDMs left untreated or LPS-primed and assessed 3 h after treatment with NHE in the absence or presence of liposomes (Lipo.). e Release of IL-1β, IL-18, and TNF, and death of WT BMDMs as treated in d . f Cryo-transmission electron microscopy analysis of liposomes left untreated ( n = 760) or assessed 1 h after treatment with NHE ( n = 800). Quantification of membrane pores of liposomes. Arrowheads indicate pores f . Scale bar, 50 nm f . NS, not significant, ** P

    Journal: Nature Communications

    Article Title: Bacillus cereus non-haemolytic enterotoxin activates the NLRP3 inflammasome

    doi: 10.1038/s41467-020-14534-3

    Figure Lengend Snippet: NHE forms pores in the cell membrane. a Schematic of toxin-induced liposomal rupture and leakage of dye. b Colorimetric analysis of liposomes left untreated, sonicated for 5 mins at 100 amplitude (CTRL) or assessed 30 mins after treatment with individual NHE subunits (C, B, or A), heat-inactivated NHE (Heat), NHE, HBL, buffer, or bovine serum albumin (BSA). c The absorbance (OD) of residual dye following treatment as in b . d Immunoblot analysis of caspase-1, gasdermin D, and IL-1β of WT BMDMs left untreated or LPS-primed and assessed 3 h after treatment with NHE in the absence or presence of liposomes (Lipo.). e Release of IL-1β, IL-18, and TNF, and death of WT BMDMs as treated in d . f Cryo-transmission electron microscopy analysis of liposomes left untreated ( n = 760) or assessed 1 h after treatment with NHE ( n = 800). Quantification of membrane pores of liposomes. Arrowheads indicate pores f . Scale bar, 50 nm f . NS, not significant, ** P

    Article Snippet: To activate the canonical NLRP3 inflammasome, BMDMs were primed with 500 ng/ml ultrapure LPS from E. coli (ALX-581-014-L002, Enzo Life Sciences) for 4 h and stimulated with 5 mM ATP (10127531001, Roche) for 45 min. To activate the NLRC4 inflammasome, BMDMs were infected with S .

    Techniques: Sonication, Transmission Assay, Electron Microscopy

    A secreted factor of B. cereus activates the inflammasome independently of HBL. a Immunoblot analysis of pro-caspase-1 (Casp-1), the active caspase-1 p20 subunit, pro-pyroptotic effector protein gasdermin D (GSDMD), the active GSDMD p30 subunit of WT BMDMs left untreated [Medium alone (Med.)] or LPS-primed and assessed either 3 h or 20 h after stimulation with the supernatant of WT B. cereus ( B. cer .) or Δ Hbl B. cereus (Δ Hbl ) (top; Supernatant (Sup.)) or infection with B. cer . or Δ Hbl (bottom; m.o.i. of 5). Release of IL-1β b , IL-18 c , and cell death d of BMDMs as treated in a . NS, not significant, *** P

    Journal: Nature Communications

    Article Title: Bacillus cereus non-haemolytic enterotoxin activates the NLRP3 inflammasome

    doi: 10.1038/s41467-020-14534-3

    Figure Lengend Snippet: A secreted factor of B. cereus activates the inflammasome independently of HBL. a Immunoblot analysis of pro-caspase-1 (Casp-1), the active caspase-1 p20 subunit, pro-pyroptotic effector protein gasdermin D (GSDMD), the active GSDMD p30 subunit of WT BMDMs left untreated [Medium alone (Med.)] or LPS-primed and assessed either 3 h or 20 h after stimulation with the supernatant of WT B. cereus ( B. cer .) or Δ Hbl B. cereus (Δ Hbl ) (top; Supernatant (Sup.)) or infection with B. cer . or Δ Hbl (bottom; m.o.i. of 5). Release of IL-1β b , IL-18 c , and cell death d of BMDMs as treated in a . NS, not significant, *** P

    Article Snippet: To activate the canonical NLRP3 inflammasome, BMDMs were primed with 500 ng/ml ultrapure LPS from E. coli (ALX-581-014-L002, Enzo Life Sciences) for 4 h and stimulated with 5 mM ATP (10127531001, Roche) for 45 min. To activate the NLRC4 inflammasome, BMDMs were infected with S .

    Techniques: Infection

    The secreted factor of B. cereus activates the NLRP3 inflammasome. a , Immunoblot analysis of caspase-1, gasdermin D, and GAPDH (loading control) in WT or mutant BMDMs left untreated or LPS-primed and assessed 20 h after stimulation with Δ Hbl supernatant, or after infection with Δ Hbl (m.o.i. of 5). b Release of IL-1β, IL-18, and TNF and death of BMDMs after treatment as in a . c Brightfield microscopy analysis of BMDMs 8 h after stimulation as in a . d Immunofluorescent analysis of ASC speck formation (red) in BMDMs after treatment as in a . Quantification of the prevalence of ASC specks is shown on the right as a percentage of total cells (DAPI). At least 200 cells from each genotype were assessed. Scale bar, 25 μm c , d . Arrowheads indicate pyroptotic cells c or ASC specks d . NS, not significant, **** P

    Journal: Nature Communications

    Article Title: Bacillus cereus non-haemolytic enterotoxin activates the NLRP3 inflammasome

    doi: 10.1038/s41467-020-14534-3

    Figure Lengend Snippet: The secreted factor of B. cereus activates the NLRP3 inflammasome. a , Immunoblot analysis of caspase-1, gasdermin D, and GAPDH (loading control) in WT or mutant BMDMs left untreated or LPS-primed and assessed 20 h after stimulation with Δ Hbl supernatant, or after infection with Δ Hbl (m.o.i. of 5). b Release of IL-1β, IL-18, and TNF and death of BMDMs after treatment as in a . c Brightfield microscopy analysis of BMDMs 8 h after stimulation as in a . d Immunofluorescent analysis of ASC speck formation (red) in BMDMs after treatment as in a . Quantification of the prevalence of ASC specks is shown on the right as a percentage of total cells (DAPI). At least 200 cells from each genotype were assessed. Scale bar, 25 μm c , d . Arrowheads indicate pyroptotic cells c or ASC specks d . NS, not significant, **** P

    Article Snippet: To activate the canonical NLRP3 inflammasome, BMDMs were primed with 500 ng/ml ultrapure LPS from E. coli (ALX-581-014-L002, Enzo Life Sciences) for 4 h and stimulated with 5 mM ATP (10127531001, Roche) for 45 min. To activate the NLRC4 inflammasome, BMDMs were infected with S .

    Techniques: Mutagenesis, Infection, Microscopy

    NHE activates the NLRP3 inflammasome. a Immunoblot analysis of caspase-1 of WT or Nlrp3 −/− BMDMs left untreated (Med.) or LPS-primed and assessed 3 h after treatment with recombinant NHE, or with the individual NHE subunits; b release of IL-1β and IL-18, and death of WT or Nlrp3 −/− BMDMs as treated in a . c , Immunofluorescent ASC speck analysis (red) in WT or Nlrp3 −/− BMDMs left untreated or LPS-primed and assessed 3 h after stimulation with NHE or 5 h after transfection with poly(dA:dT). Quantification of ASC specks as a percentage of total cells (DAPI). At least 200 cells from each genotype were assessed. Arrowheads indicate ASC specks. d Scanning electron microscopy analysis (top) or transmission electron microscopy analysis (bottom) of WT BMDMs left untreated or LPS-primed and assessed 3 h after treatment with NHE. Scale bar, 12 μm c , 2 μm d . NS, not significant, *** P

    Journal: Nature Communications

    Article Title: Bacillus cereus non-haemolytic enterotoxin activates the NLRP3 inflammasome

    doi: 10.1038/s41467-020-14534-3

    Figure Lengend Snippet: NHE activates the NLRP3 inflammasome. a Immunoblot analysis of caspase-1 of WT or Nlrp3 −/− BMDMs left untreated (Med.) or LPS-primed and assessed 3 h after treatment with recombinant NHE, or with the individual NHE subunits; b release of IL-1β and IL-18, and death of WT or Nlrp3 −/− BMDMs as treated in a . c , Immunofluorescent ASC speck analysis (red) in WT or Nlrp3 −/− BMDMs left untreated or LPS-primed and assessed 3 h after stimulation with NHE or 5 h after transfection with poly(dA:dT). Quantification of ASC specks as a percentage of total cells (DAPI). At least 200 cells from each genotype were assessed. Arrowheads indicate ASC specks. d Scanning electron microscopy analysis (top) or transmission electron microscopy analysis (bottom) of WT BMDMs left untreated or LPS-primed and assessed 3 h after treatment with NHE. Scale bar, 12 μm c , 2 μm d . NS, not significant, *** P

    Article Snippet: To activate the canonical NLRP3 inflammasome, BMDMs were primed with 500 ng/ml ultrapure LPS from E. coli (ALX-581-014-L002, Enzo Life Sciences) for 4 h and stimulated with 5 mM ATP (10127531001, Roche) for 45 min. To activate the NLRC4 inflammasome, BMDMs were infected with S .

    Techniques: Recombinant, Transfection, Electron Microscopy, Transmission Assay

    Neutralization of both HBL and NHE abrogates inflammasome activation. a Immunoblot analysis of caspase-1 and gasdermin D, of WT BMDMs left untreated or LPS-primed and assessed 20 h after treatment with the supernatant of WT B. cereus (Sup.) treated with an isotype control (Iso.), anti-HBL-neutralizing antibodies (α-HBL), anti-NHE-neutralizing antibodies (α-NHE) or with both anti-HBL- and anti-NHE-neutralizing antibodies (α-Tox.). Release of IL-1β b , and IL-18 c of BMDMs, death of BMDMs d , and release of TNF e , of BMDMs as treated in a . f Immunoblot analysis of caspase-1 and gasdermin D of WT BMDMs left untreated or LPS-primed and assessed 20 h after treatment with the supernatant of Δ Hbl treated with an isotype control or anti-NHE-neutralizing antibodies or assessed 20 h after treatment with the supernatant of Δ Nhe B. cereus (Δ Nhe Sup.) treated with an isotype control or anti-HBL-neutralizing antibodies. Release of IL-1β g , and IL-18 h of BMDMs, death of BMDMs i , and release of TNF j of BMDMs as treated in f . NS, not significant, ** P

    Journal: Nature Communications

    Article Title: Bacillus cereus non-haemolytic enterotoxin activates the NLRP3 inflammasome

    doi: 10.1038/s41467-020-14534-3

    Figure Lengend Snippet: Neutralization of both HBL and NHE abrogates inflammasome activation. a Immunoblot analysis of caspase-1 and gasdermin D, of WT BMDMs left untreated or LPS-primed and assessed 20 h after treatment with the supernatant of WT B. cereus (Sup.) treated with an isotype control (Iso.), anti-HBL-neutralizing antibodies (α-HBL), anti-NHE-neutralizing antibodies (α-NHE) or with both anti-HBL- and anti-NHE-neutralizing antibodies (α-Tox.). Release of IL-1β b , and IL-18 c of BMDMs, death of BMDMs d , and release of TNF e , of BMDMs as treated in a . f Immunoblot analysis of caspase-1 and gasdermin D of WT BMDMs left untreated or LPS-primed and assessed 20 h after treatment with the supernatant of Δ Hbl treated with an isotype control or anti-NHE-neutralizing antibodies or assessed 20 h after treatment with the supernatant of Δ Nhe B. cereus (Δ Nhe Sup.) treated with an isotype control or anti-HBL-neutralizing antibodies. Release of IL-1β g , and IL-18 h of BMDMs, death of BMDMs i , and release of TNF j of BMDMs as treated in f . NS, not significant, ** P

    Article Snippet: To activate the canonical NLRP3 inflammasome, BMDMs were primed with 500 ng/ml ultrapure LPS from E. coli (ALX-581-014-L002, Enzo Life Sciences) for 4 h and stimulated with 5 mM ATP (10127531001, Roche) for 45 min. To activate the NLRC4 inflammasome, BMDMs were infected with S .

    Techniques: Neutralization, Activation Assay

    Antagonistic effects of the activators of TLRs, PI-3K/Akt, ERK1/2, p38/JNK, and NF-κB pathways on the anti-IAV activity of OMT. A549 cells were infected with IAV (MOI = 0.001), treated with or without ribavirin (25 μg/mL) and OMT (50 μg/mL), and simultaneously treated with or without TLR3 activator (polyI:C, 100 ng/mL), TLR4 activator LPS-B5 (1 μg/mL), TLR7/8 activator R-848 (10 μg/mL), PI-3K/Akt activator (IGF-1, 100 ng/mL), ERK activator (EGF, 100 ng/mL), p38/JNK activator (Anisomycin, 10 μM), and NF-κB activator (PMA, 1 µg/mL). The test doses of all activators were determined according to previous reports and our preliminary tests. After 48 h, cell viability was determined by the SRB method ( A ) and IAV vRNA levels were determined by a qRT-PCR assay ( B ). Data shown were mean ± SD of three independent experiments performed in triplicate. * p

    Journal: International Journal of Molecular Sciences

    Article Title: Oxymatrine Inhibits Influenza A Virus Replication and Inflammation via TLR4, p38 MAPK and NF-κB Pathways

    doi: 10.3390/ijms19040965

    Figure Lengend Snippet: Antagonistic effects of the activators of TLRs, PI-3K/Akt, ERK1/2, p38/JNK, and NF-κB pathways on the anti-IAV activity of OMT. A549 cells were infected with IAV (MOI = 0.001), treated with or without ribavirin (25 μg/mL) and OMT (50 μg/mL), and simultaneously treated with or without TLR3 activator (polyI:C, 100 ng/mL), TLR4 activator LPS-B5 (1 μg/mL), TLR7/8 activator R-848 (10 μg/mL), PI-3K/Akt activator (IGF-1, 100 ng/mL), ERK activator (EGF, 100 ng/mL), p38/JNK activator (Anisomycin, 10 μM), and NF-κB activator (PMA, 1 µg/mL). The test doses of all activators were determined according to previous reports and our preliminary tests. After 48 h, cell viability was determined by the SRB method ( A ) and IAV vRNA levels were determined by a qRT-PCR assay ( B ). Data shown were mean ± SD of three independent experiments performed in triplicate. * p

    Article Snippet: Poly(I:C) (tlrl-pic), LPS-B5 (tlrl-pb5lps), R-848 (tlrl-r848) and PMA (tlrl-pma) were purchased from InvivoGen (San Diego, CA, USA).

    Techniques: Activity Assay, Infection, Sulforhodamine B Assay, Quantitative RT-PCR

    Determination of effect of lithium on nitric oxide production on LPS-induced Raw 264.7 cells. Cells were seeded at 6 × 10 6 cell/ml and treated with lithium (5 and 10 mM), 10 mM NaCl, and 100 ng/ml LPS, combining each of the chemicals with LPS for 24 hrs. Cells were stained with DAF2-DA and Hoechst for 20 min at RT. The measurement of the fluorescence was accomplished by using fluorescent inverted Nikon Ti-E microscope at 20x magnification (b). Fluorescence intensity of the pictures was measured using the NIS Element view imaging software (Nikon, Japan) (a). Graphs were executed using GraphPad Prism 4 software and the statistical analysis was carried out using GraphPad InStat software and ANOVA (Tukey–Kramer) ( ∗ p

    Journal: International Journal of Inflammation

    Article Title: The Effect of Lithium on Inflammation-Associated Genes in Lipopolysaccharide-Activated Raw 264.7 Macrophages

    doi: 10.1155/2020/8340195

    Figure Lengend Snippet: Determination of effect of lithium on nitric oxide production on LPS-induced Raw 264.7 cells. Cells were seeded at 6 × 10 6 cell/ml and treated with lithium (5 and 10 mM), 10 mM NaCl, and 100 ng/ml LPS, combining each of the chemicals with LPS for 24 hrs. Cells were stained with DAF2-DA and Hoechst for 20 min at RT. The measurement of the fluorescence was accomplished by using fluorescent inverted Nikon Ti-E microscope at 20x magnification (b). Fluorescence intensity of the pictures was measured using the NIS Element view imaging software (Nikon, Japan) (a). Graphs were executed using GraphPad Prism 4 software and the statistical analysis was carried out using GraphPad InStat software and ANOVA (Tukey–Kramer) ( ∗ p

    Article Snippet: Therefore, in this study, cells were seeded on coverslips in 6-well plates overnight and then pretreated with LiCl (5 and 10 mM), 10 mM NaCl and activated with 100 ng/ml Ultrapure LPS (Invitrogen, USA) for 24 hrs.

    Techniques: Staining, Fluorescence, Microscopy, Imaging, Software

    Evaluation of the effects of lithium on nuclear translocation of NF- κ B after LPS activation of Raw 264.7 cells. Cells were cultured at a density of 2 × 10 5 cells/ml on coverslips and treated with 10 mM lithium, 10 mM NaCl, 100 ng/ml ultrapure LPS, and a combination of salts and LPS for 90 min. Cells were then fixed with 3.7% paraformaldehyde and thereafter semipermeabilised with 0.1% Triton-X100 in 1% BSA for 30 min. The nonspecific binding was blocked for an hour with 1% BSA and then anti-NF- κ B antibody (1 : 500) was added for an hour, and then the cells were washed 3x with 1x PBS and stained for 30 min with anti-rabbit IgG-FITC (1 : 2000). After 30 min incubation, cells were washed, followed by DNA staining with 25 μ g/ml Hoechst for 20 min. Thereafter, coverslips were mounted on glass slides with mounting medium and then NF-kB was analysed and photographed under fluorescence inverted microscope (Nikon Eclipse TS100F, Japan).

    Journal: International Journal of Inflammation

    Article Title: The Effect of Lithium on Inflammation-Associated Genes in Lipopolysaccharide-Activated Raw 264.7 Macrophages

    doi: 10.1155/2020/8340195

    Figure Lengend Snippet: Evaluation of the effects of lithium on nuclear translocation of NF- κ B after LPS activation of Raw 264.7 cells. Cells were cultured at a density of 2 × 10 5 cells/ml on coverslips and treated with 10 mM lithium, 10 mM NaCl, 100 ng/ml ultrapure LPS, and a combination of salts and LPS for 90 min. Cells were then fixed with 3.7% paraformaldehyde and thereafter semipermeabilised with 0.1% Triton-X100 in 1% BSA for 30 min. The nonspecific binding was blocked for an hour with 1% BSA and then anti-NF- κ B antibody (1 : 500) was added for an hour, and then the cells were washed 3x with 1x PBS and stained for 30 min with anti-rabbit IgG-FITC (1 : 2000). After 30 min incubation, cells were washed, followed by DNA staining with 25 μ g/ml Hoechst for 20 min. Thereafter, coverslips were mounted on glass slides with mounting medium and then NF-kB was analysed and photographed under fluorescence inverted microscope (Nikon Eclipse TS100F, Japan).

    Article Snippet: Therefore, in this study, cells were seeded on coverslips in 6-well plates overnight and then pretreated with LiCl (5 and 10 mM), 10 mM NaCl and activated with 100 ng/ml Ultrapure LPS (Invitrogen, USA) for 24 hrs.

    Techniques: Translocation Assay, Activation Assay, Cell Culture, Binding Assay, Staining, Incubation, Fluorescence, Inverted Microscopy

    Effect of lithium on nitric oxide (NO) production assessed using Griess reagent on LPS-activated Raw 264.7 cells. Cells were seeded at a density of 6 × 10 6 cells/ml and treatment was executed by pretreating with 10 mM NaCl and lithium (1.25, 2.5, 5, and 10 mM) for an hour thereafter; other set of wells that are already treated with lithium and NaCl were stimulated with 10 μ g/ml LPS for 24 hrs. Quantitation of NO production was carried out by GloMax-Multi microplate at 550 nm (Promega, USA). Treatments that encompass various concentrations of lithium and 10 mM NaCl combined with LPS were compared with LPS activated wells. Graphs were executed using GraphPad Prism 4 software and the statistical analysis was carried out using GraphPad InStat software and ANOVA (Tukey–Kramer) ( ∗ p

    Journal: International Journal of Inflammation

    Article Title: The Effect of Lithium on Inflammation-Associated Genes in Lipopolysaccharide-Activated Raw 264.7 Macrophages

    doi: 10.1155/2020/8340195

    Figure Lengend Snippet: Effect of lithium on nitric oxide (NO) production assessed using Griess reagent on LPS-activated Raw 264.7 cells. Cells were seeded at a density of 6 × 10 6 cells/ml and treatment was executed by pretreating with 10 mM NaCl and lithium (1.25, 2.5, 5, and 10 mM) for an hour thereafter; other set of wells that are already treated with lithium and NaCl were stimulated with 10 μ g/ml LPS for 24 hrs. Quantitation of NO production was carried out by GloMax-Multi microplate at 550 nm (Promega, USA). Treatments that encompass various concentrations of lithium and 10 mM NaCl combined with LPS were compared with LPS activated wells. Graphs were executed using GraphPad Prism 4 software and the statistical analysis was carried out using GraphPad InStat software and ANOVA (Tukey–Kramer) ( ∗ p

    Article Snippet: Therefore, in this study, cells were seeded on coverslips in 6-well plates overnight and then pretreated with LiCl (5 and 10 mM), 10 mM NaCl and activated with 100 ng/ml Ultrapure LPS (Invitrogen, USA) for 24 hrs.

    Techniques: Quantitation Assay, Software

    Western blot analysis of the levels of TLR4 (A, n = 6/group; B, n = 6–8/group), IBA-1 (C, n = 4–7/group; D, n = 7–8/group), and GFAP (E, n = 6–8/group; F, n = 10–11/group) proteins in the rat ipsilateral dorsal lumbar spinal cord (A, C, E) and DRG (B, D, F) after repeated ith. administration of LPS-RSU (20 µg/5 µL, ith. ) on day 7 after chronic constriction injury (CCI). The data are presented as the means ± SEM. Inter-group differences were analyzed using one-way ANOVA followed by Bonferroni’s multiple comparisons test. ∗ p

    Journal: Pharmaceutical Biology

    Article Title: Lipopolysaccharide from Rhodobacter sphaeroides (TLR4 antagonist) attenuates hypersensitivity and modulates nociceptive factors

    doi: 10.1080/13880209.2018.1457061

    Figure Lengend Snippet: Western blot analysis of the levels of TLR4 (A, n = 6/group; B, n = 6–8/group), IBA-1 (C, n = 4–7/group; D, n = 7–8/group), and GFAP (E, n = 6–8/group; F, n = 10–11/group) proteins in the rat ipsilateral dorsal lumbar spinal cord (A, C, E) and DRG (B, D, F) after repeated ith. administration of LPS-RSU (20 µg/5 µL, ith. ) on day 7 after chronic constriction injury (CCI). The data are presented as the means ± SEM. Inter-group differences were analyzed using one-way ANOVA followed by Bonferroni’s multiple comparisons test. ∗ p

    Article Snippet: Materials and methods: Behavioural (von Frey’s/cold plate) tests were performed with Wistar male rats after intrathecal administration of a TLR4 antagonist (LPS-RS ULTRAPURE (LPS-RSU), 20 μG: lipopolysaccharide from Rhodobacter sphaeroides , InvivoGen, San Diego, CA) 16 H and 1 h before chronic constriction injury (cci) to the sciatic nerve and then daily for 7 d. three groups were used: an intact group and two cci-exposed groups that received vehicle or LPS-RSU. tissue [spinal cord/dorsal root ganglia (DRG)] for western blot analysis was collected on day 7.

    Techniques: Western Blot

    ), and pre-emptive repeated administration of the TLR4 antagonist LPS-RSU potentiates the increase in the number of IBA-1-positive cells in the DRG after chronic constriction injury (CCI). Moreover, LPS-RSU induces a change in the ratio between IL-18/IL18BP and MMP-9/TIMP-1, in favour of the antinociceptive neuropathic factors IL-18BP and TIMP-1. Additionally, LPS-RSU administration increased the IL-6 level, which under some circumstances is known to have analgesic properties. In summary, pharmacological blockade of TLR4 diminished hypersensitivity and modulated the levels of nociceptive proteins.

    Journal: Pharmaceutical Biology

    Article Title: Lipopolysaccharide from Rhodobacter sphaeroides (TLR4 antagonist) attenuates hypersensitivity and modulates nociceptive factors

    doi: 10.1080/13880209.2018.1457061

    Figure Lengend Snippet: ), and pre-emptive repeated administration of the TLR4 antagonist LPS-RSU potentiates the increase in the number of IBA-1-positive cells in the DRG after chronic constriction injury (CCI). Moreover, LPS-RSU induces a change in the ratio between IL-18/IL18BP and MMP-9/TIMP-1, in favour of the antinociceptive neuropathic factors IL-18BP and TIMP-1. Additionally, LPS-RSU administration increased the IL-6 level, which under some circumstances is known to have analgesic properties. In summary, pharmacological blockade of TLR4 diminished hypersensitivity and modulated the levels of nociceptive proteins.

    Article Snippet: Materials and methods: Behavioural (von Frey’s/cold plate) tests were performed with Wistar male rats after intrathecal administration of a TLR4 antagonist (LPS-RS ULTRAPURE (LPS-RSU), 20 μG: lipopolysaccharide from Rhodobacter sphaeroides , InvivoGen, San Diego, CA) 16 H and 1 h before chronic constriction injury (cci) to the sciatic nerve and then daily for 7 d. three groups were used: an intact group and two cci-exposed groups that received vehicle or LPS-RSU. tissue [spinal cord/dorsal root ganglia (DRG)] for western blot analysis was collected on day 7.

    Techniques:

    Phosphorylation of S5 inhibits NLRP3 activation. (A–C) Quantification of IL-1β and TNF by ELISA in NLRP3-deficient iMOs reconstituted with NLRP3-mCitrine WT or indicated mutations after 3 h LPS priming (for [A] TNF) and stimulated with (B) lethal toxin (6 h) or (C) nigericin (1 h). (A–C) n = 3 ± SEM; (C) *, P

    Journal: The Journal of Experimental Medicine

    Article Title: NLRP3 inflammasome assembly is regulated by phosphorylation of the pyrin domain

    doi: 10.1084/jem.20160933

    Figure Lengend Snippet: Phosphorylation of S5 inhibits NLRP3 activation. (A–C) Quantification of IL-1β and TNF by ELISA in NLRP3-deficient iMOs reconstituted with NLRP3-mCitrine WT or indicated mutations after 3 h LPS priming (for [A] TNF) and stimulated with (B) lethal toxin (6 h) or (C) nigericin (1 h). (A–C) n = 3 ± SEM; (C) *, P

    Article Snippet: Reagents Ultrapure LPS was obtained from InvivoGen, nigericin was obtained from Invitrogen, okadaic acid was obtained from Calbiochem, lethal factor and protective antigen were obtained from List Biological Laboratories, and Gene Juice was purchased from Merck.

    Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay

    Phosphorylation interferes with a polybasic cluster in the NLRP3 PYD. (A) Electrostatic surface representation of NLRP3 PYD. (left) Nonphosphorylated PYD (PDB accession no. 3QF2); (right) surface charge changes with phosphorylated serine 5 modeled. (B) Mutations introduced in the N/C terminal region of NLRP3 PYD. (C–E) Quantification of IL-1β and TNF by ELISA in NLRP3 deficient iMOs reconstituted with NLRP3-mCitrine WT or PYD-N-terminal (R7A, K9A, and R12A) or PYD-C-terminal (K86A, R89A, and K93A) mutations after 3 h LPS priming (for [C] TNF) and stimulated with (D) lethal toxin (6 h) or (E) nigericin (1 h). (C–E), n = 3 ± SEM. (F) Immunoblot of NLRP3-deficient iMOs reconstituted with NLRP3-mCitrine WT or indicated mutations after 2 h LPS priming and left untreated (none) or stimulated with nigericin (1 h). Immunoblots are representative of two independent experiments.

    Journal: The Journal of Experimental Medicine

    Article Title: NLRP3 inflammasome assembly is regulated by phosphorylation of the pyrin domain

    doi: 10.1084/jem.20160933

    Figure Lengend Snippet: Phosphorylation interferes with a polybasic cluster in the NLRP3 PYD. (A) Electrostatic surface representation of NLRP3 PYD. (left) Nonphosphorylated PYD (PDB accession no. 3QF2); (right) surface charge changes with phosphorylated serine 5 modeled. (B) Mutations introduced in the N/C terminal region of NLRP3 PYD. (C–E) Quantification of IL-1β and TNF by ELISA in NLRP3 deficient iMOs reconstituted with NLRP3-mCitrine WT or PYD-N-terminal (R7A, K9A, and R12A) or PYD-C-terminal (K86A, R89A, and K93A) mutations after 3 h LPS priming (for [C] TNF) and stimulated with (D) lethal toxin (6 h) or (E) nigericin (1 h). (C–E), n = 3 ± SEM. (F) Immunoblot of NLRP3-deficient iMOs reconstituted with NLRP3-mCitrine WT or indicated mutations after 2 h LPS priming and left untreated (none) or stimulated with nigericin (1 h). Immunoblots are representative of two independent experiments.

    Article Snippet: Reagents Ultrapure LPS was obtained from InvivoGen, nigericin was obtained from Invitrogen, okadaic acid was obtained from Calbiochem, lethal factor and protective antigen were obtained from List Biological Laboratories, and Gene Juice was purchased from Merck.

    Techniques: Enzyme-linked Immunosorbent Assay, Western Blot

    NLRP3 PYD gets dephosphorylated with involvement of protein phosphatase 2A. (A and B) Quantification of (A) TNF and (B) IL-1β by ELISA in NLRP3-deficient iMOs reconstituted with NLRP3-FLAG after 15 min pretreatment with okadaic acid (OKA) or left untreated (none), followed by 2 h LPS priming (for TNF) and stimulated with nigericin (1 h). (A and B) n = 2 ± SEM. (C) Ion intensities for the peptide containing the phosphorylated S3 (acTphSVRCKL; m/z = 493.23046; z = 2), normalized to the nonphosphorylated counterpart (acTSVRCKL; m/z = 453.24729; z = 2) are plotted at the indicated retention times for LPS-treated and LPS+OKA-treated samples. Representative of two independent experiments. (D) Ratio of the apex of phosphorylated to nonphosphorylated peptide intensities (mean ± SEM of replicates, representative of two independent experiments). (E) Immunoblot of NLRP3-deficient iMOs reconstituted with NLRP3-FLAG WT and transfected with the indicated siRNAs for 40 h. Immunoblots are representative of three independent experiments. (F and G) Same as E, but showing mRNA levels (fold of control, normalized to Hprt expression) for (F) Ppp2ca and (G) Ppp2cb. (F and G) n = 4 ± SEM. ***, P

    Journal: The Journal of Experimental Medicine

    Article Title: NLRP3 inflammasome assembly is regulated by phosphorylation of the pyrin domain

    doi: 10.1084/jem.20160933

    Figure Lengend Snippet: NLRP3 PYD gets dephosphorylated with involvement of protein phosphatase 2A. (A and B) Quantification of (A) TNF and (B) IL-1β by ELISA in NLRP3-deficient iMOs reconstituted with NLRP3-FLAG after 15 min pretreatment with okadaic acid (OKA) or left untreated (none), followed by 2 h LPS priming (for TNF) and stimulated with nigericin (1 h). (A and B) n = 2 ± SEM. (C) Ion intensities for the peptide containing the phosphorylated S3 (acTphSVRCKL; m/z = 493.23046; z = 2), normalized to the nonphosphorylated counterpart (acTSVRCKL; m/z = 453.24729; z = 2) are plotted at the indicated retention times for LPS-treated and LPS+OKA-treated samples. Representative of two independent experiments. (D) Ratio of the apex of phosphorylated to nonphosphorylated peptide intensities (mean ± SEM of replicates, representative of two independent experiments). (E) Immunoblot of NLRP3-deficient iMOs reconstituted with NLRP3-FLAG WT and transfected with the indicated siRNAs for 40 h. Immunoblots are representative of three independent experiments. (F and G) Same as E, but showing mRNA levels (fold of control, normalized to Hprt expression) for (F) Ppp2ca and (G) Ppp2cb. (F and G) n = 4 ± SEM. ***, P

    Article Snippet: Reagents Ultrapure LPS was obtained from InvivoGen, nigericin was obtained from Invitrogen, okadaic acid was obtained from Calbiochem, lethal factor and protective antigen were obtained from List Biological Laboratories, and Gene Juice was purchased from Merck.

    Techniques: Enzyme-linked Immunosorbent Assay, Transfection, Western Blot, Expressing

    Effect of 25(OH)D 3 and 1,25(OH) 2 D 3 on the production of pro-inflammatory mediators by hPdLF with silenced VDR. Cells were transfected with either VDR siRNA or control siRNA and stimulated with P. gingivalis LPS ( Pg LPS, 1 µg/ml) or heat-killed P. gingivalis (hk Pg , 10 8 cells/ml) for 24 h in the presence or in the absence of 25(OH)D 3 or 1,25(OH) 2 D 3 . # means significantly different from control group (2 −▵▵Ct = 1). * means significantly different from cells stimulated with either P. gingivalis LPS or heat-killed P. gingivalis only

    Journal: PLoS ONE

    Article Title: Both 25-Hydroxyvitamin-D3 and 1,25-Dihydroxyvitamin-D3 Reduces Inflammatory Response in Human Periodontal Ligament Cells

    doi: 10.1371/journal.pone.0090301

    Figure Lengend Snippet: Effect of 25(OH)D 3 and 1,25(OH) 2 D 3 on the production of pro-inflammatory mediators by hPdLF with silenced VDR. Cells were transfected with either VDR siRNA or control siRNA and stimulated with P. gingivalis LPS ( Pg LPS, 1 µg/ml) or heat-killed P. gingivalis (hk Pg , 10 8 cells/ml) for 24 h in the presence or in the absence of 25(OH)D 3 or 1,25(OH) 2 D 3 . # means significantly different from control group (2 −▵▵Ct = 1). * means significantly different from cells stimulated with either P. gingivalis LPS or heat-killed P. gingivalis only

    Article Snippet: Commercially available ultrapure P. gingivalis LPS, heat-killed P. gingivalis , and ultrapure E. coli LPS (all from Invivogen, San Diego, USA) were used in the present study.

    Techniques: Transfection

    Effect of 25(OH)D 3 and 1,25(OH) 2 D 3 on the gene-expression levels of pro-inflammatory mediators in primary hPdLC in response to stimulation with P. gingivalis LPS or heat-killed P. gingivalis . Cells were stimulated with P. gingivalis LPS ( Pg LPS, 1 µg/ml) or heat-killed P. gingivalis (hk Pg , 10 8 cells/ml) for 24 h in the presence or in the absence of different concentrations of 25(OH)D 3 or 1,25(OH) 2 D 3 . Gene expression levels of IL-6 (A), IL-8 (B), and MCP-1 (C) were measured using q-PCR. Y-axes represent the n-fold expression levels of target gene in relation to non-stimulated cells (control). Data are presented as mean±SEM of six different donors. # means significantly different from control group (2 −▵▵Ct = 1). * means significantly different from cells stimulated with P. gingivalis LPS or heat-killed P. gingivalis only.

    Journal: PLoS ONE

    Article Title: Both 25-Hydroxyvitamin-D3 and 1,25-Dihydroxyvitamin-D3 Reduces Inflammatory Response in Human Periodontal Ligament Cells

    doi: 10.1371/journal.pone.0090301

    Figure Lengend Snippet: Effect of 25(OH)D 3 and 1,25(OH) 2 D 3 on the gene-expression levels of pro-inflammatory mediators in primary hPdLC in response to stimulation with P. gingivalis LPS or heat-killed P. gingivalis . Cells were stimulated with P. gingivalis LPS ( Pg LPS, 1 µg/ml) or heat-killed P. gingivalis (hk Pg , 10 8 cells/ml) for 24 h in the presence or in the absence of different concentrations of 25(OH)D 3 or 1,25(OH) 2 D 3 . Gene expression levels of IL-6 (A), IL-8 (B), and MCP-1 (C) were measured using q-PCR. Y-axes represent the n-fold expression levels of target gene in relation to non-stimulated cells (control). Data are presented as mean±SEM of six different donors. # means significantly different from control group (2 −▵▵Ct = 1). * means significantly different from cells stimulated with P. gingivalis LPS or heat-killed P. gingivalis only.

    Article Snippet: Commercially available ultrapure P. gingivalis LPS, heat-killed P. gingivalis , and ultrapure E. coli LPS (all from Invivogen, San Diego, USA) were used in the present study.

    Techniques: Expressing, Polymerase Chain Reaction

    Effect of 25(OH)D 3 and 1,25(OH) 2 D 3 on the production of pro-inflammatory mediators by hPdLF in response to stimulation with P. gingivalis LPS. Cells were stimulated with P. gingivalis LPS ( Pg LPS, 1 µg/ml) for 24 h in the presence or in the absence of different concentrations of 25(OH)D 3 or 1,25(OH) 2 D 3 . The levels of IL-6 (A), IL-8 (B), and MCP-1 (C) were measured in cell supernatants using ELISA. # means significantly different from control group (non stimulated cells). * means significantly different from group stimulated with P.gingivalis LPS only

    Journal: PLoS ONE

    Article Title: Both 25-Hydroxyvitamin-D3 and 1,25-Dihydroxyvitamin-D3 Reduces Inflammatory Response in Human Periodontal Ligament Cells

    doi: 10.1371/journal.pone.0090301

    Figure Lengend Snippet: Effect of 25(OH)D 3 and 1,25(OH) 2 D 3 on the production of pro-inflammatory mediators by hPdLF in response to stimulation with P. gingivalis LPS. Cells were stimulated with P. gingivalis LPS ( Pg LPS, 1 µg/ml) for 24 h in the presence or in the absence of different concentrations of 25(OH)D 3 or 1,25(OH) 2 D 3 . The levels of IL-6 (A), IL-8 (B), and MCP-1 (C) were measured in cell supernatants using ELISA. # means significantly different from control group (non stimulated cells). * means significantly different from group stimulated with P.gingivalis LPS only

    Article Snippet: Commercially available ultrapure P. gingivalis LPS, heat-killed P. gingivalis , and ultrapure E. coli LPS (all from Invivogen, San Diego, USA) were used in the present study.

    Techniques: Enzyme-linked Immunosorbent Assay

    Effect of 25(OH)D 3 and 1,25(OH) 2 D 3 on the gene-expression levels of pro-inflammatory mediators in hPdLF with silenced VDR in response to stimulation with P. gingivalis LPS or heat-killed P. gingivalis . Gene expression levels of IL-6, IL-8, and MCP-1 were measured using q-PCR in hPdLF after transfection with either VDR siRNA or control siRNA and stimulation with P. gingivalis LPS (A, Pg LPS, 1 µg/ml) or heat-killed P. gingivalis (B, hk Pg , 10 8 cells/ml) in the presence or in the absence of 25(OH)D 3 or 1,25(OH) 2 D 3 . Y-axes represent the n-fold expression levels of target gene in relation to non-stimulated cells. # means significantly different from control group (2 −▵▵Ct = 1). * means significantly different from cells stimulated with heat-killed P. gingivalis LPS or heat-killed P. gingivalis only.

    Journal: PLoS ONE

    Article Title: Both 25-Hydroxyvitamin-D3 and 1,25-Dihydroxyvitamin-D3 Reduces Inflammatory Response in Human Periodontal Ligament Cells

    doi: 10.1371/journal.pone.0090301

    Figure Lengend Snippet: Effect of 25(OH)D 3 and 1,25(OH) 2 D 3 on the gene-expression levels of pro-inflammatory mediators in hPdLF with silenced VDR in response to stimulation with P. gingivalis LPS or heat-killed P. gingivalis . Gene expression levels of IL-6, IL-8, and MCP-1 were measured using q-PCR in hPdLF after transfection with either VDR siRNA or control siRNA and stimulation with P. gingivalis LPS (A, Pg LPS, 1 µg/ml) or heat-killed P. gingivalis (B, hk Pg , 10 8 cells/ml) in the presence or in the absence of 25(OH)D 3 or 1,25(OH) 2 D 3 . Y-axes represent the n-fold expression levels of target gene in relation to non-stimulated cells. # means significantly different from control group (2 −▵▵Ct = 1). * means significantly different from cells stimulated with heat-killed P. gingivalis LPS or heat-killed P. gingivalis only.

    Article Snippet: Commercially available ultrapure P. gingivalis LPS, heat-killed P. gingivalis , and ultrapure E. coli LPS (all from Invivogen, San Diego, USA) were used in the present study.

    Techniques: Expressing, Polymerase Chain Reaction, Transfection

    Effect of 25(OH)D 3 and 1,25(OH) 2 D 3 on the production of pro-inflammatory mediators by primary hPdLC in response to stimulation with P. gingivalis LPS or heat-killed P. gingivalis . Cells were stimulated with P. gingivalis LPS ( Pg LPS, 1 µg/ml) or heat-killed P. gingivalis (hk Pg , 10 8 cells/ml) for 24 h in the presence or in the absence of different concentrations of 25(OH)D 3 or 1,25(OH) 2 D 3 . The levels of IL-6 (A), IL-8 (B), and MCP-1 (C) were measured in cell supernatants using ELISA. # means significantly different from control group (non stimulated cells). * means significantly different from group stimulated with P.gingivalis LPS or heat-killed P. gingivalis only.

    Journal: PLoS ONE

    Article Title: Both 25-Hydroxyvitamin-D3 and 1,25-Dihydroxyvitamin-D3 Reduces Inflammatory Response in Human Periodontal Ligament Cells

    doi: 10.1371/journal.pone.0090301

    Figure Lengend Snippet: Effect of 25(OH)D 3 and 1,25(OH) 2 D 3 on the production of pro-inflammatory mediators by primary hPdLC in response to stimulation with P. gingivalis LPS or heat-killed P. gingivalis . Cells were stimulated with P. gingivalis LPS ( Pg LPS, 1 µg/ml) or heat-killed P. gingivalis (hk Pg , 10 8 cells/ml) for 24 h in the presence or in the absence of different concentrations of 25(OH)D 3 or 1,25(OH) 2 D 3 . The levels of IL-6 (A), IL-8 (B), and MCP-1 (C) were measured in cell supernatants using ELISA. # means significantly different from control group (non stimulated cells). * means significantly different from group stimulated with P.gingivalis LPS or heat-killed P. gingivalis only.

    Article Snippet: Commercially available ultrapure P. gingivalis LPS, heat-killed P. gingivalis , and ultrapure E. coli LPS (all from Invivogen, San Diego, USA) were used in the present study.

    Techniques: Enzyme-linked Immunosorbent Assay

    Gene expression levels of pro-inflammatory mediators in hPdLF in response to stimulation with E. coli LPS, P. gingivalis LPS, and heat-killed P. gingivalis . Cells were stimulated with E. coli LPS (1 µg/ml), P. gingivalis LPS (0.1–1 µg/ml), or heat-killed P. gingivalis (10 7 –10 8 cells/ml) for 24 h. Gene expression levels of IL-6 (A), IL-8 (B), and MCP-1 (C) were measured using q-PCR. Y-axes represent the n-fold expression levels of target gene in relation to non-stimulated cells (control).

    Journal: PLoS ONE

    Article Title: Both 25-Hydroxyvitamin-D3 and 1,25-Dihydroxyvitamin-D3 Reduces Inflammatory Response in Human Periodontal Ligament Cells

    doi: 10.1371/journal.pone.0090301

    Figure Lengend Snippet: Gene expression levels of pro-inflammatory mediators in hPdLF in response to stimulation with E. coli LPS, P. gingivalis LPS, and heat-killed P. gingivalis . Cells were stimulated with E. coli LPS (1 µg/ml), P. gingivalis LPS (0.1–1 µg/ml), or heat-killed P. gingivalis (10 7 –10 8 cells/ml) for 24 h. Gene expression levels of IL-6 (A), IL-8 (B), and MCP-1 (C) were measured using q-PCR. Y-axes represent the n-fold expression levels of target gene in relation to non-stimulated cells (control).

    Article Snippet: Commercially available ultrapure P. gingivalis LPS, heat-killed P. gingivalis , and ultrapure E. coli LPS (all from Invivogen, San Diego, USA) were used in the present study.

    Techniques: Expressing, Polymerase Chain Reaction

    Effect of 25(OH)D 3 and 1,25(OH) 2 D 3 on the gene-expression levels of pro-inflammatory mediators in hPdLF in response to stimulation with P. gingivalis LPS. Cells were stimulated with P. gingivalis LPS ( Pg LPS, 1 µg/ml) for 24 h in the presence or in the absence of different concentrations of 25(OH)D 3 or 1,25(OH) 2 D 3 . Gene expression levels of IL-6 (A), IL-8 (B), and MCP-1 (C) were measured using q-PCR. Y-axes represent the n-fold expression levels of target gene in relation to non-stimulated cells (control). # means significantly different from control group (2 −▵▵Ct = 1). * means significantly different from cells stimulated with P. gingivalis LPS only.

    Journal: PLoS ONE

    Article Title: Both 25-Hydroxyvitamin-D3 and 1,25-Dihydroxyvitamin-D3 Reduces Inflammatory Response in Human Periodontal Ligament Cells

    doi: 10.1371/journal.pone.0090301

    Figure Lengend Snippet: Effect of 25(OH)D 3 and 1,25(OH) 2 D 3 on the gene-expression levels of pro-inflammatory mediators in hPdLF in response to stimulation with P. gingivalis LPS. Cells were stimulated with P. gingivalis LPS ( Pg LPS, 1 µg/ml) for 24 h in the presence or in the absence of different concentrations of 25(OH)D 3 or 1,25(OH) 2 D 3 . Gene expression levels of IL-6 (A), IL-8 (B), and MCP-1 (C) were measured using q-PCR. Y-axes represent the n-fold expression levels of target gene in relation to non-stimulated cells (control). # means significantly different from control group (2 −▵▵Ct = 1). * means significantly different from cells stimulated with P. gingivalis LPS only.

    Article Snippet: Commercially available ultrapure P. gingivalis LPS, heat-killed P. gingivalis , and ultrapure E. coli LPS (all from Invivogen, San Diego, USA) were used in the present study.

    Techniques: Expressing, Polymerase Chain Reaction

    HIV infection increases cell-surface expression of TLR4 and CD14 in KCs. (A) Representative FACS analyses of TLR4 expression before (gray histogram) and after (white histogram) HIV infection and LPS treatment. (B) FACS demonstrating quantitative expression level of TLR4 and CD68 in KCs derived from 8 patients directly after isolation and with adherence enrichment and HIV infection KCs. (C) Cumulative data on the TLR4 expression level in CD68-positive cells after HIV infection ( n = 8; P = 0.04). (D) Representative FACS analyses of CD14 expression before (gray histogram) and after (white histogram) HIV infection and LPS treatment. (E) FACS demonstrating quantitative expression level of CD14 and CD68 in KCs derived from 8 patients directly after isolation and with adherence enrichment and HIV infection. (F) Cumulative data on the CD14 expression level in CD68-positive cells after HIV infection ( n = 8; P = 0.0038).

    Journal: Journal of Leukocyte Biology

    Article Title: Frontline Science: HIV infection of Kupffer cells results in an amplified proinflammatory response to LPS

    doi: 10.1189/jlb.3HI0516-242R

    Figure Lengend Snippet: HIV infection increases cell-surface expression of TLR4 and CD14 in KCs. (A) Representative FACS analyses of TLR4 expression before (gray histogram) and after (white histogram) HIV infection and LPS treatment. (B) FACS demonstrating quantitative expression level of TLR4 and CD68 in KCs derived from 8 patients directly after isolation and with adherence enrichment and HIV infection KCs. (C) Cumulative data on the TLR4 expression level in CD68-positive cells after HIV infection ( n = 8; P = 0.04). (D) Representative FACS analyses of CD14 expression before (gray histogram) and after (white histogram) HIV infection and LPS treatment. (E) FACS demonstrating quantitative expression level of CD14 and CD68 in KCs derived from 8 patients directly after isolation and with adherence enrichment and HIV infection. (F) Cumulative data on the CD14 expression level in CD68-positive cells after HIV infection ( n = 8; P = 0.0038).

    Article Snippet: 72 h post-HIV-1BaL -infection KCs were challenged with TLR4 ligand ultrapure LPS (TLR-pb5LPS; InvivoGen, San Diego, CA, USA) at a dose of 100 ng/ml for 4 h, followed by RNA extraction and RT-qPCR.

    Techniques: Infection, Expressing, FACS, Derivative Assay, Isolation