caspase-1 production primed thp-1 cells Search Results


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    Thermo Fisher gene exp pdgfb mm01298578 m1
    Gene Exp Pdgfb Mm01298578 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 98/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore rna
    AOSD NETs are potent triggers of NLRP3 inflammasomes. After stimulation with neutrophil extracellular trap <t>(NET)</t> DNA from adult-onset Still’s disease (AOSD) and healthy controls (HC), mitochondrial DNA (mtDNA), genomic DNA g(DNA), and <t>RNA,</t> the expression ( a ) and secretion ( b ) of interleukin (IL)-1β and IL-18 in THP-1 monocytes were measured using RT-PCR and ELISA, respectively. c Representative immunoblot analysis for NLRP3 inflammasomes in THP-1 monocytes. The images are representative of three independent Western blot experiments. d After stimulation with NET DNA and non-NET source nucleic acids shown above, the expression of IL-1β and IL-18 in PBMC-derived CD14 + monocytes from healthy controls was measured using RT-PCR. e The expression of IL-1β, pro-IL-1β, and NLRP3 in cell lysates (LYS) and IL-1β in supernatants (SN) from THP-1 monocytes after stimulation with NET-DNA in the presence of the NLRP3 inhibitor MCC950 and DNase I was measured by Western blot. The images are representative of three independent Western blot experiments. The histograms show the means ± SD. * P
    Rna, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 5994 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Adipogen nlrp3
    AOSD NETs are potent triggers of NLRP3 inflammasomes. After stimulation with neutrophil extracellular trap <t>(NET)</t> DNA from adult-onset Still’s disease (AOSD) and healthy controls (HC), mitochondrial DNA (mtDNA), genomic DNA g(DNA), and <t>RNA,</t> the expression ( a ) and secretion ( b ) of interleukin (IL)-1β and IL-18 in THP-1 monocytes were measured using RT-PCR and ELISA, respectively. c Representative immunoblot analysis for NLRP3 inflammasomes in THP-1 monocytes. The images are representative of three independent Western blot experiments. d After stimulation with NET DNA and non-NET source nucleic acids shown above, the expression of IL-1β and IL-18 in PBMC-derived CD14 + monocytes from healthy controls was measured using RT-PCR. e The expression of IL-1β, pro-IL-1β, and NLRP3 in cell lysates (LYS) and IL-1β in supernatants (SN) from THP-1 monocytes after stimulation with NET-DNA in the presence of the NLRP3 inhibitor MCC950 and DNase I was measured by Western blot. The images are representative of three independent Western blot experiments. The histograms show the means ± SD. * P
    Nlrp3, supplied by Adipogen, used in various techniques. Bioz Stars score: 93/100, based on 1361 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    InvivoGen msu crystals
    AOSD NETs are potent triggers of NLRP3 inflammasomes. After stimulation with neutrophil extracellular trap <t>(NET)</t> DNA from adult-onset Still’s disease (AOSD) and healthy controls (HC), mitochondrial DNA (mtDNA), genomic DNA g(DNA), and <t>RNA,</t> the expression ( a ) and secretion ( b ) of interleukin (IL)-1β and IL-18 in THP-1 monocytes were measured using RT-PCR and ELISA, respectively. c Representative immunoblot analysis for NLRP3 inflammasomes in THP-1 monocytes. The images are representative of three independent Western blot experiments. d After stimulation with NET DNA and non-NET source nucleic acids shown above, the expression of IL-1β and IL-18 in PBMC-derived CD14 + monocytes from healthy controls was measured using RT-PCR. e The expression of IL-1β, pro-IL-1β, and NLRP3 in cell lysates (LYS) and IL-1β in supernatants (SN) from THP-1 monocytes after stimulation with NET-DNA in the presence of the NLRP3 inhibitor MCC950 and DNase I was measured by Western blot. The images are representative of three independent Western blot experiments. The histograms show the means ± SD. * P
    Msu Crystals, supplied by InvivoGen, used in various techniques. Bioz Stars score: 99/100, based on 105 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti p53 polyclonal antibody pab
    AOSD NETs are potent triggers of NLRP3 inflammasomes. After stimulation with neutrophil extracellular trap <t>(NET)</t> DNA from adult-onset Still’s disease (AOSD) and healthy controls (HC), mitochondrial DNA (mtDNA), genomic DNA g(DNA), and <t>RNA,</t> the expression ( a ) and secretion ( b ) of interleukin (IL)-1β and IL-18 in THP-1 monocytes were measured using RT-PCR and ELISA, respectively. c Representative immunoblot analysis for NLRP3 inflammasomes in THP-1 monocytes. The images are representative of three independent Western blot experiments. d After stimulation with NET DNA and non-NET source nucleic acids shown above, the expression of IL-1β and IL-18 in PBMC-derived CD14 + monocytes from healthy controls was measured using RT-PCR. e The expression of IL-1β, pro-IL-1β, and NLRP3 in cell lysates (LYS) and IL-1β in supernatants (SN) from THP-1 monocytes after stimulation with NET-DNA in the presence of the NLRP3 inhibitor MCC950 and DNase I was measured by Western blot. The images are representative of three independent Western blot experiments. The histograms show the means ± SD. * P
    Anti P53 Polyclonal Antibody Pab, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    InvivoGen nigericin
    Subcellular location of NLRP3 inflammasome requires F-actin but not active polymerization. ( A,B ) Primed THP-1 cells were activated with ( A ) ATP and ( B ) <t>nigericin</t> after pretreatment with cytochalasin D (CytoD) or latrunculin B (LatB). Cytosolic (CYT) and cytoskeletal (CSK) fractions were analyzed by Western blot. The cropped blots were run under the same experimental conditions; Data are representative of 3 independent experiments. ( C ) Confocal microscopy of subcellular location of ASC and NLRP3 in primed THP-1 cells treated or not with cytochalasin D (CytoD) and latrunculin B (LatB) prior to activation with ATP or nigericin for 6 h; Blue, nuclei. Outlined areas are enlarged in top right corners. Scale bars, 10 μm. Representative pictures of 3 independent experiments.
    Nigericin, supplied by InvivoGen, used in various techniques. Bioz Stars score: 99/100, based on 357 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher caspase 1
    F-actin downregulates NLRP3 inflammasome activity. ( A ) Actin polymerization was performed with 9.6 μM of pyrene-labeled G-actin containing ATP- or nigericin-activated THP-1 cells lysate. The polymerization was initiated by addition of actin polymerization buffer (arrow). Representative experiments out of 3 are presented. ( B ) Area under the curve (AUC) represented in ( A ) was calculated using GraphPad Prism version 6. Data are means ± SEM of at least 3 independent. ( C,D ) IL-1β production in culture supernatants of primed THP-1 cells pretreated with increasing doses of cytochalasin D (CytoD) or latrunculin B (LatB) and then activated by ATP ( C ) and nigericin ( D ) for 6 h. Data are means ± SEM of at least 3 independent. ( E,F ) IL-1β production in culture supernatants of LPS-primed primary human monocytes pretreated with increasing doses of cytochalasin D (CytoD) or latrunculin B (LatB) and stimulated or not with ATP ( E ) or nigericin ( F ) for 15 min. Data are means ± SEM of at least 3 independent. ( G,H ) <t>Caspase-1</t> in supernatants (SN) of THP-1 cells treated as indicated and assessed by ELISA (upper panels) and Western blot (bottom panels); total, uncleaved caspase-1 in cell lysate is presented in bottom panels. The cropped blots were run under the same experimental conditions. The asterisk indicates nonspecific crossreactive bands. Data are means ± SEM of at least 4 independent experiments (upper panels) or representative of 3 independent experiments (bottom panels). Statistical significance was determined by Mann-Whitney U analysis. See also Figures S1 and S2 .
    Caspase 1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 335 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher opti mem medium
    F-actin downregulates NLRP3 inflammasome activity. ( A ) Actin polymerization was performed with 9.6 μM of pyrene-labeled G-actin containing ATP- or nigericin-activated THP-1 cells lysate. The polymerization was initiated by addition of actin polymerization buffer (arrow). Representative experiments out of 3 are presented. ( B ) Area under the curve (AUC) represented in ( A ) was calculated using GraphPad Prism version 6. Data are means ± SEM of at least 3 independent. ( C,D ) IL-1β production in culture supernatants of primed THP-1 cells pretreated with increasing doses of cytochalasin D (CytoD) or latrunculin B (LatB) and then activated by ATP ( C ) and nigericin ( D ) for 6 h. Data are means ± SEM of at least 3 independent. ( E,F ) IL-1β production in culture supernatants of LPS-primed primary human monocytes pretreated with increasing doses of cytochalasin D (CytoD) or latrunculin B (LatB) and stimulated or not with ATP ( E ) or nigericin ( F ) for 15 min. Data are means ± SEM of at least 3 independent. ( G,H ) <t>Caspase-1</t> in supernatants (SN) of THP-1 cells treated as indicated and assessed by ELISA (upper panels) and Western blot (bottom panels); total, uncleaved caspase-1 in cell lysate is presented in bottom panels. The cropped blots were run under the same experimental conditions. The asterisk indicates nonspecific crossreactive bands. Data are means ± SEM of at least 4 independent experiments (upper panels) or representative of 3 independent experiments (bottom panels). Statistical significance was determined by Mann-Whitney U analysis. See also Figures S1 and S2 .
    Opti Mem Medium, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 11755 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    InvivoGen p gingivalis
    F-actin downregulates NLRP3 inflammasome activity. ( A ) Actin polymerization was performed with 9.6 μM of pyrene-labeled G-actin containing ATP- or nigericin-activated THP-1 cells lysate. The polymerization was initiated by addition of actin polymerization buffer (arrow). Representative experiments out of 3 are presented. ( B ) Area under the curve (AUC) represented in ( A ) was calculated using GraphPad Prism version 6. Data are means ± SEM of at least 3 independent. ( C,D ) IL-1β production in culture supernatants of primed THP-1 cells pretreated with increasing doses of cytochalasin D (CytoD) or latrunculin B (LatB) and then activated by ATP ( C ) and nigericin ( D ) for 6 h. Data are means ± SEM of at least 3 independent. ( E,F ) IL-1β production in culture supernatants of LPS-primed primary human monocytes pretreated with increasing doses of cytochalasin D (CytoD) or latrunculin B (LatB) and stimulated or not with ATP ( E ) or nigericin ( F ) for 15 min. Data are means ± SEM of at least 3 independent. ( G,H ) <t>Caspase-1</t> in supernatants (SN) of THP-1 cells treated as indicated and assessed by ELISA (upper panels) and Western blot (bottom panels); total, uncleaved caspase-1 in cell lysate is presented in bottom panels. The cropped blots were run under the same experimental conditions. The asterisk indicates nonspecific crossreactive bands. Data are means ± SEM of at least 4 independent experiments (upper panels) or representative of 3 independent experiments (bottom panels). Statistical significance was determined by Mann-Whitney U analysis. See also Figures S1 and S2 .
    P Gingivalis, supplied by InvivoGen, used in various techniques. Bioz Stars score: 92/100, based on 347 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    AOSD NETs are potent triggers of NLRP3 inflammasomes. After stimulation with neutrophil extracellular trap (NET) DNA from adult-onset Still’s disease (AOSD) and healthy controls (HC), mitochondrial DNA (mtDNA), genomic DNA g(DNA), and RNA, the expression ( a ) and secretion ( b ) of interleukin (IL)-1β and IL-18 in THP-1 monocytes were measured using RT-PCR and ELISA, respectively. c Representative immunoblot analysis for NLRP3 inflammasomes in THP-1 monocytes. The images are representative of three independent Western blot experiments. d After stimulation with NET DNA and non-NET source nucleic acids shown above, the expression of IL-1β and IL-18 in PBMC-derived CD14 + monocytes from healthy controls was measured using RT-PCR. e The expression of IL-1β, pro-IL-1β, and NLRP3 in cell lysates (LYS) and IL-1β in supernatants (SN) from THP-1 monocytes after stimulation with NET-DNA in the presence of the NLRP3 inhibitor MCC950 and DNase I was measured by Western blot. The images are representative of three independent Western blot experiments. The histograms show the means ± SD. * P

    Journal: Arthritis Research & Therapy

    Article Title: Increased neutrophil extracellular traps activate NLRP3 and inflammatory macrophages in adult-onset Still’s disease

    doi: 10.1186/s13075-018-1800-z

    Figure Lengend Snippet: AOSD NETs are potent triggers of NLRP3 inflammasomes. After stimulation with neutrophil extracellular trap (NET) DNA from adult-onset Still’s disease (AOSD) and healthy controls (HC), mitochondrial DNA (mtDNA), genomic DNA g(DNA), and RNA, the expression ( a ) and secretion ( b ) of interleukin (IL)-1β and IL-18 in THP-1 monocytes were measured using RT-PCR and ELISA, respectively. c Representative immunoblot analysis for NLRP3 inflammasomes in THP-1 monocytes. The images are representative of three independent Western blot experiments. d After stimulation with NET DNA and non-NET source nucleic acids shown above, the expression of IL-1β and IL-18 in PBMC-derived CD14 + monocytes from healthy controls was measured using RT-PCR. e The expression of IL-1β, pro-IL-1β, and NLRP3 in cell lysates (LYS) and IL-1β in supernatants (SN) from THP-1 monocytes after stimulation with NET-DNA in the presence of the NLRP3 inhibitor MCC950 and DNase I was measured by Western blot. The images are representative of three independent Western blot experiments. The histograms show the means ± SD. * P

    Article Snippet: Media were subsequently removed and replaced with phenol red-free, serum-free RPMI prior to treatment with 250 ng NET DNA, mtDNA, genomic DNA, RNA, or 5 mM ATP (Sigma, St. Louis, MO) for 2 h. mtDNA, genomic DNA, and RNA were isolated from 293 T cells according to previously published methods [ ].

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Western Blot, Derivative Assay

    Subcellular location of NLRP3 inflammasome requires F-actin but not active polymerization. ( A,B ) Primed THP-1 cells were activated with ( A ) ATP and ( B ) nigericin after pretreatment with cytochalasin D (CytoD) or latrunculin B (LatB). Cytosolic (CYT) and cytoskeletal (CSK) fractions were analyzed by Western blot. The cropped blots were run under the same experimental conditions; Data are representative of 3 independent experiments. ( C ) Confocal microscopy of subcellular location of ASC and NLRP3 in primed THP-1 cells treated or not with cytochalasin D (CytoD) and latrunculin B (LatB) prior to activation with ATP or nigericin for 6 h; Blue, nuclei. Outlined areas are enlarged in top right corners. Scale bars, 10 μm. Representative pictures of 3 independent experiments.

    Journal: Scientific Reports

    Article Title: F-actin dampens NLRP3 inflammasome activity via Flightless-I and LRRFIP2

    doi: 10.1038/srep29834

    Figure Lengend Snippet: Subcellular location of NLRP3 inflammasome requires F-actin but not active polymerization. ( A,B ) Primed THP-1 cells were activated with ( A ) ATP and ( B ) nigericin after pretreatment with cytochalasin D (CytoD) or latrunculin B (LatB). Cytosolic (CYT) and cytoskeletal (CSK) fractions were analyzed by Western blot. The cropped blots were run under the same experimental conditions; Data are representative of 3 independent experiments. ( C ) Confocal microscopy of subcellular location of ASC and NLRP3 in primed THP-1 cells treated or not with cytochalasin D (CytoD) and latrunculin B (LatB) prior to activation with ATP or nigericin for 6 h; Blue, nuclei. Outlined areas are enlarged in top right corners. Scale bars, 10 μm. Representative pictures of 3 independent experiments.

    Article Snippet: IL-1β and caspase-1 production Primed THP-1 cells were treated for 45 min with the indicated inhibitor in Opti-MEM medium (Life Technology) prior to activation by 5 mM ATP, 1 μM nigericin, 500 μg/ml MSU crystals, 5 μg/ml Poly(dA:dT)/LyoVec™ (Invivogen) or 3 μg FLA-PA (transfected with ViaFect) for 6 h (MSU crystals were a kind gift of Dr. A. Scanu, University of Padova).

    Techniques: Western Blot, Confocal Microscopy, Activation Assay

    F-actin downregulates NLRP3 inflammasome activity. ( A ) Actin polymerization was performed with 9.6 μM of pyrene-labeled G-actin containing ATP- or nigericin-activated THP-1 cells lysate. The polymerization was initiated by addition of actin polymerization buffer (arrow). Representative experiments out of 3 are presented. ( B ) Area under the curve (AUC) represented in ( A ) was calculated using GraphPad Prism version 6. Data are means ± SEM of at least 3 independent. ( C,D ) IL-1β production in culture supernatants of primed THP-1 cells pretreated with increasing doses of cytochalasin D (CytoD) or latrunculin B (LatB) and then activated by ATP ( C ) and nigericin ( D ) for 6 h. Data are means ± SEM of at least 3 independent. ( E,F ) IL-1β production in culture supernatants of LPS-primed primary human monocytes pretreated with increasing doses of cytochalasin D (CytoD) or latrunculin B (LatB) and stimulated or not with ATP ( E ) or nigericin ( F ) for 15 min. Data are means ± SEM of at least 3 independent. ( G,H ) Caspase-1 in supernatants (SN) of THP-1 cells treated as indicated and assessed by ELISA (upper panels) and Western blot (bottom panels); total, uncleaved caspase-1 in cell lysate is presented in bottom panels. The cropped blots were run under the same experimental conditions. The asterisk indicates nonspecific crossreactive bands. Data are means ± SEM of at least 4 independent experiments (upper panels) or representative of 3 independent experiments (bottom panels). Statistical significance was determined by Mann-Whitney U analysis. See also Figures S1 and S2 .

    Journal: Scientific Reports

    Article Title: F-actin dampens NLRP3 inflammasome activity via Flightless-I and LRRFIP2

    doi: 10.1038/srep29834

    Figure Lengend Snippet: F-actin downregulates NLRP3 inflammasome activity. ( A ) Actin polymerization was performed with 9.6 μM of pyrene-labeled G-actin containing ATP- or nigericin-activated THP-1 cells lysate. The polymerization was initiated by addition of actin polymerization buffer (arrow). Representative experiments out of 3 are presented. ( B ) Area under the curve (AUC) represented in ( A ) was calculated using GraphPad Prism version 6. Data are means ± SEM of at least 3 independent. ( C,D ) IL-1β production in culture supernatants of primed THP-1 cells pretreated with increasing doses of cytochalasin D (CytoD) or latrunculin B (LatB) and then activated by ATP ( C ) and nigericin ( D ) for 6 h. Data are means ± SEM of at least 3 independent. ( E,F ) IL-1β production in culture supernatants of LPS-primed primary human monocytes pretreated with increasing doses of cytochalasin D (CytoD) or latrunculin B (LatB) and stimulated or not with ATP ( E ) or nigericin ( F ) for 15 min. Data are means ± SEM of at least 3 independent. ( G,H ) Caspase-1 in supernatants (SN) of THP-1 cells treated as indicated and assessed by ELISA (upper panels) and Western blot (bottom panels); total, uncleaved caspase-1 in cell lysate is presented in bottom panels. The cropped blots were run under the same experimental conditions. The asterisk indicates nonspecific crossreactive bands. Data are means ± SEM of at least 4 independent experiments (upper panels) or representative of 3 independent experiments (bottom panels). Statistical significance was determined by Mann-Whitney U analysis. See also Figures S1 and S2 .

    Article Snippet: IL-1β and caspase-1 production Primed THP-1 cells were treated for 45 min with the indicated inhibitor in Opti-MEM medium (Life Technology) prior to activation by 5 mM ATP, 1 μM nigericin, 500 μg/ml MSU crystals, 5 μg/ml Poly(dA:dT)/LyoVec™ (Invivogen) or 3 μg FLA-PA (transfected with ViaFect) for 6 h (MSU crystals were a kind gift of Dr. A. Scanu, University of Padova).

    Techniques: Activity Assay, Labeling, Enzyme-linked Immunosorbent Assay, Western Blot, MANN-WHITNEY

    Ca 2+ increase the NLRP3 inflammasome activation by enhancing the severing of F-actin by FliI. ( A ) IL-1β production in culture supernatants of primed THP-1 cells pretreated with increasing doses of CaCl 2 (Ca 2+ ) and stimulated with nigericin. Data are means ± SEM of at least 3 independent. ( B ) Caspase-1 in supernatants (SN) of THP-1 cells pretreated with 1.6 mM CaCl 2 and activated by nigericin as indicated and assessed by ELISA. Data are means ± SEM of at least 3 independent. ( C ) Actin depolymerization was performed with 0.2 μg/ml of pyrene-labeled F-actin containing nigericin-activated THP-1 cells lysate. Representative pictures of 3 independent experiments. ( D ) Area under the curve (AUC) represented in ( C ) was calculated using GraphPad Prism version 6. Data are represented as mean ± SEM of at least 3 independent experiments. Statistical significance was determined by Mann-Whitney U analysis. See Figure S4 .

    Journal: Scientific Reports

    Article Title: F-actin dampens NLRP3 inflammasome activity via Flightless-I and LRRFIP2

    doi: 10.1038/srep29834

    Figure Lengend Snippet: Ca 2+ increase the NLRP3 inflammasome activation by enhancing the severing of F-actin by FliI. ( A ) IL-1β production in culture supernatants of primed THP-1 cells pretreated with increasing doses of CaCl 2 (Ca 2+ ) and stimulated with nigericin. Data are means ± SEM of at least 3 independent. ( B ) Caspase-1 in supernatants (SN) of THP-1 cells pretreated with 1.6 mM CaCl 2 and activated by nigericin as indicated and assessed by ELISA. Data are means ± SEM of at least 3 independent. ( C ) Actin depolymerization was performed with 0.2 μg/ml of pyrene-labeled F-actin containing nigericin-activated THP-1 cells lysate. Representative pictures of 3 independent experiments. ( D ) Area under the curve (AUC) represented in ( C ) was calculated using GraphPad Prism version 6. Data are represented as mean ± SEM of at least 3 independent experiments. Statistical significance was determined by Mann-Whitney U analysis. See Figure S4 .

    Article Snippet: IL-1β and caspase-1 production Primed THP-1 cells were treated for 45 min with the indicated inhibitor in Opti-MEM medium (Life Technology) prior to activation by 5 mM ATP, 1 μM nigericin, 500 μg/ml MSU crystals, 5 μg/ml Poly(dA:dT)/LyoVec™ (Invivogen) or 3 μg FLA-PA (transfected with ViaFect) for 6 h (MSU crystals were a kind gift of Dr. A. Scanu, University of Padova).

    Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Labeling, MANN-WHITNEY

    FliI and LRRFIP2 enable inhibition of NLRP3 inflammasome activity via co-localization with F-actin. ( A ) Confocal microscopy of ATP- and nigericin-activated THP-1 cells. Blue, nuclei. Outlined areas are enlarged in top right corners. Scale bars, 10 μm. Representative pictures of 3 independent experiments. ( B ) Western blot analysis of FliI and LRRFIP2 expression in THP-1 cells stably transduced with lentivirus carrying FliI and LRRFIP2 shRNA. Data are representative of 3 independent experiments. ( C ) Confocal microscopy of ATP- and nigericin-activated THP-1 cells transduced with FliI and LRRFIP2 shRNA. Blue, nuclei. Outlined areas are enlarged in top right corners. Scale bars, 10 μm. Representative pictures of 3 independent experiments. ( D ) IL-1β production in culture supernatants of THP-1 cells transduced with FliI and LRRFIP2 shRNA and unstimulated or stimulated with ATP and nigericin. Data are means ± SEM of at least 3 independent. ( E ) Caspase-1 in supernatants (SN) of THP-1 cells treated as indicated and assessed by ELISA (upper panels) and Western blot (bottom panels); the presence of caspase-1 was assessed by Western blot into cells lysate. Data are represented as mean ± SEM of at least 3 independent experiments or representative of 3 independent experiments. Statistical significance was determined by Mann-Whitney U analysis. The cropped blots were run under the same experimental conditions. See Figure S3 .

    Journal: Scientific Reports

    Article Title: F-actin dampens NLRP3 inflammasome activity via Flightless-I and LRRFIP2

    doi: 10.1038/srep29834

    Figure Lengend Snippet: FliI and LRRFIP2 enable inhibition of NLRP3 inflammasome activity via co-localization with F-actin. ( A ) Confocal microscopy of ATP- and nigericin-activated THP-1 cells. Blue, nuclei. Outlined areas are enlarged in top right corners. Scale bars, 10 μm. Representative pictures of 3 independent experiments. ( B ) Western blot analysis of FliI and LRRFIP2 expression in THP-1 cells stably transduced with lentivirus carrying FliI and LRRFIP2 shRNA. Data are representative of 3 independent experiments. ( C ) Confocal microscopy of ATP- and nigericin-activated THP-1 cells transduced with FliI and LRRFIP2 shRNA. Blue, nuclei. Outlined areas are enlarged in top right corners. Scale bars, 10 μm. Representative pictures of 3 independent experiments. ( D ) IL-1β production in culture supernatants of THP-1 cells transduced with FliI and LRRFIP2 shRNA and unstimulated or stimulated with ATP and nigericin. Data are means ± SEM of at least 3 independent. ( E ) Caspase-1 in supernatants (SN) of THP-1 cells treated as indicated and assessed by ELISA (upper panels) and Western blot (bottom panels); the presence of caspase-1 was assessed by Western blot into cells lysate. Data are represented as mean ± SEM of at least 3 independent experiments or representative of 3 independent experiments. Statistical significance was determined by Mann-Whitney U analysis. The cropped blots were run under the same experimental conditions. See Figure S3 .

    Article Snippet: IL-1β and caspase-1 production Primed THP-1 cells were treated for 45 min with the indicated inhibitor in Opti-MEM medium (Life Technology) prior to activation by 5 mM ATP, 1 μM nigericin, 500 μg/ml MSU crystals, 5 μg/ml Poly(dA:dT)/LyoVec™ (Invivogen) or 3 μg FLA-PA (transfected with ViaFect) for 6 h (MSU crystals were a kind gift of Dr. A. Scanu, University of Padova).

    Techniques: Inhibition, Activity Assay, Confocal Microscopy, Western Blot, Expressing, Stable Transfection, Transduction, shRNA, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

    Schematic representation of NLRP3 inflammasome regulation. TLRs activation or PMA (Signal 1) induce the expression of pro-IL-1β, pro-IL-18 and NLRP3. Signal 2 provided by ATP or nigericin induces NLRP3 inflammasome assembly through a dynein- and microtubules-dependent transport. Then, we postulate that the increase of intracellular Ca 2+ concentration through channels such as TRPM7, TRPV2 and/or InsP 3 R, enhances the ability of FliI to sever F-actin and thus abrogates LRRFIP2-FliI-dependent NLRP3 inflammasome inhibition increasing IL-1β and IL-18 production. The colored part of the picture is the present study.

    Journal: Scientific Reports

    Article Title: F-actin dampens NLRP3 inflammasome activity via Flightless-I and LRRFIP2

    doi: 10.1038/srep29834

    Figure Lengend Snippet: Schematic representation of NLRP3 inflammasome regulation. TLRs activation or PMA (Signal 1) induce the expression of pro-IL-1β, pro-IL-18 and NLRP3. Signal 2 provided by ATP or nigericin induces NLRP3 inflammasome assembly through a dynein- and microtubules-dependent transport. Then, we postulate that the increase of intracellular Ca 2+ concentration through channels such as TRPM7, TRPV2 and/or InsP 3 R, enhances the ability of FliI to sever F-actin and thus abrogates LRRFIP2-FliI-dependent NLRP3 inflammasome inhibition increasing IL-1β and IL-18 production. The colored part of the picture is the present study.

    Article Snippet: IL-1β and caspase-1 production Primed THP-1 cells were treated for 45 min with the indicated inhibitor in Opti-MEM medium (Life Technology) prior to activation by 5 mM ATP, 1 μM nigericin, 500 μg/ml MSU crystals, 5 μg/ml Poly(dA:dT)/LyoVec™ (Invivogen) or 3 μg FLA-PA (transfected with ViaFect) for 6 h (MSU crystals were a kind gift of Dr. A. Scanu, University of Padova).

    Techniques: Activation Assay, Expressing, Concentration Assay, Inhibition

    NLRP3 inflammasome interacts with F-actin in ATP- and nigericin-treated THP-1 cells. ( A,B ) Primed THP-1 cells were activated with 5 mM ATP or 1 μM nigericin (NIG) for 6 h. Cytosolic (CYT) and cytoskeletal (CSK) fractions of ( A ) ATP- and ( B ) nigericin-activated cells were subjected to Western blot and analyzed for the presence of ASC, NLRP3, pro-IL-1β and vimentin (control for cytoskeletal fraction). The cropped blots were run under the same experimental conditions; Data are representative of 3 independent experiments. ( C ) Confocal microscopy of ATP- and nigericin-activated THP-1 cells; nuclei are stained in blue. Outlined areas are enlarged in top right corners. Scale bars, 10 μm. Data are representative of 3 independent experiments. ( D ) Primed THP-1 cells were activated with 5 mM ATP or 1 μM nigericin (NIG) for 6 h, immunoprecipitated with anti-actin and subjected to Western blot and analyzed for the presence of ASC, NLRP3 and actin. The cropped blots were run under the same experimental conditions; Data are representative of 2 independent experiments.

    Journal: Scientific Reports

    Article Title: F-actin dampens NLRP3 inflammasome activity via Flightless-I and LRRFIP2

    doi: 10.1038/srep29834

    Figure Lengend Snippet: NLRP3 inflammasome interacts with F-actin in ATP- and nigericin-treated THP-1 cells. ( A,B ) Primed THP-1 cells were activated with 5 mM ATP or 1 μM nigericin (NIG) for 6 h. Cytosolic (CYT) and cytoskeletal (CSK) fractions of ( A ) ATP- and ( B ) nigericin-activated cells were subjected to Western blot and analyzed for the presence of ASC, NLRP3, pro-IL-1β and vimentin (control for cytoskeletal fraction). The cropped blots were run under the same experimental conditions; Data are representative of 3 independent experiments. ( C ) Confocal microscopy of ATP- and nigericin-activated THP-1 cells; nuclei are stained in blue. Outlined areas are enlarged in top right corners. Scale bars, 10 μm. Data are representative of 3 independent experiments. ( D ) Primed THP-1 cells were activated with 5 mM ATP or 1 μM nigericin (NIG) for 6 h, immunoprecipitated with anti-actin and subjected to Western blot and analyzed for the presence of ASC, NLRP3 and actin. The cropped blots were run under the same experimental conditions; Data are representative of 2 independent experiments.

    Article Snippet: IL-1β and caspase-1 production Primed THP-1 cells were treated for 45 min with the indicated inhibitor in Opti-MEM medium (Life Technology) prior to activation by 5 mM ATP, 1 μM nigericin, 500 μg/ml MSU crystals, 5 μg/ml Poly(dA:dT)/LyoVec™ (Invivogen) or 3 μg FLA-PA (transfected with ViaFect) for 6 h (MSU crystals were a kind gift of Dr. A. Scanu, University of Padova).

    Techniques: Western Blot, Confocal Microscopy, Staining, Immunoprecipitation

    F-actin downregulates NLRP3 inflammasome activity. ( A ) Actin polymerization was performed with 9.6 μM of pyrene-labeled G-actin containing ATP- or nigericin-activated THP-1 cells lysate. The polymerization was initiated by addition of actin polymerization buffer (arrow). Representative experiments out of 3 are presented. ( B ) Area under the curve (AUC) represented in ( A ) was calculated using GraphPad Prism version 6. Data are means ± SEM of at least 3 independent. ( C,D ) IL-1β production in culture supernatants of primed THP-1 cells pretreated with increasing doses of cytochalasin D (CytoD) or latrunculin B (LatB) and then activated by ATP ( C ) and nigericin ( D ) for 6 h. Data are means ± SEM of at least 3 independent. ( E,F ) IL-1β production in culture supernatants of LPS-primed primary human monocytes pretreated with increasing doses of cytochalasin D (CytoD) or latrunculin B (LatB) and stimulated or not with ATP ( E ) or nigericin ( F ) for 15 min. Data are means ± SEM of at least 3 independent. ( G,H ) Caspase-1 in supernatants (SN) of THP-1 cells treated as indicated and assessed by ELISA (upper panels) and Western blot (bottom panels); total, uncleaved caspase-1 in cell lysate is presented in bottom panels. The cropped blots were run under the same experimental conditions. The asterisk indicates nonspecific crossreactive bands. Data are means ± SEM of at least 4 independent experiments (upper panels) or representative of 3 independent experiments (bottom panels). Statistical significance was determined by Mann-Whitney U analysis. See also Figures S1 and S2 .

    Journal: Scientific Reports

    Article Title: F-actin dampens NLRP3 inflammasome activity via Flightless-I and LRRFIP2

    doi: 10.1038/srep29834

    Figure Lengend Snippet: F-actin downregulates NLRP3 inflammasome activity. ( A ) Actin polymerization was performed with 9.6 μM of pyrene-labeled G-actin containing ATP- or nigericin-activated THP-1 cells lysate. The polymerization was initiated by addition of actin polymerization buffer (arrow). Representative experiments out of 3 are presented. ( B ) Area under the curve (AUC) represented in ( A ) was calculated using GraphPad Prism version 6. Data are means ± SEM of at least 3 independent. ( C,D ) IL-1β production in culture supernatants of primed THP-1 cells pretreated with increasing doses of cytochalasin D (CytoD) or latrunculin B (LatB) and then activated by ATP ( C ) and nigericin ( D ) for 6 h. Data are means ± SEM of at least 3 independent. ( E,F ) IL-1β production in culture supernatants of LPS-primed primary human monocytes pretreated with increasing doses of cytochalasin D (CytoD) or latrunculin B (LatB) and stimulated or not with ATP ( E ) or nigericin ( F ) for 15 min. Data are means ± SEM of at least 3 independent. ( G,H ) Caspase-1 in supernatants (SN) of THP-1 cells treated as indicated and assessed by ELISA (upper panels) and Western blot (bottom panels); total, uncleaved caspase-1 in cell lysate is presented in bottom panels. The cropped blots were run under the same experimental conditions. The asterisk indicates nonspecific crossreactive bands. Data are means ± SEM of at least 4 independent experiments (upper panels) or representative of 3 independent experiments (bottom panels). Statistical significance was determined by Mann-Whitney U analysis. See also Figures S1 and S2 .

    Article Snippet: Culture supernatants were tested for the production of IL-1β or caspase-1 by enzyme immunoassay (Human IL-1β, eBioscience, San Diego, CA, USA and Caspase-1, Quantikine; R & D Systems, Minneapolis, MN, USA).

    Techniques: Activity Assay, Labeling, Enzyme-linked Immunosorbent Assay, Western Blot, MANN-WHITNEY

    Ca 2+ increase the NLRP3 inflammasome activation by enhancing the severing of F-actin by FliI. ( A ) IL-1β production in culture supernatants of primed THP-1 cells pretreated with increasing doses of CaCl 2 (Ca 2+ ) and stimulated with nigericin. Data are means ± SEM of at least 3 independent. ( B ) Caspase-1 in supernatants (SN) of THP-1 cells pretreated with 1.6 mM CaCl 2 and activated by nigericin as indicated and assessed by ELISA. Data are means ± SEM of at least 3 independent. ( C ) Actin depolymerization was performed with 0.2 μg/ml of pyrene-labeled F-actin containing nigericin-activated THP-1 cells lysate. Representative pictures of 3 independent experiments. ( D ) Area under the curve (AUC) represented in ( C ) was calculated using GraphPad Prism version 6. Data are represented as mean ± SEM of at least 3 independent experiments. Statistical significance was determined by Mann-Whitney U analysis. See Figure S4 .

    Journal: Scientific Reports

    Article Title: F-actin dampens NLRP3 inflammasome activity via Flightless-I and LRRFIP2

    doi: 10.1038/srep29834

    Figure Lengend Snippet: Ca 2+ increase the NLRP3 inflammasome activation by enhancing the severing of F-actin by FliI. ( A ) IL-1β production in culture supernatants of primed THP-1 cells pretreated with increasing doses of CaCl 2 (Ca 2+ ) and stimulated with nigericin. Data are means ± SEM of at least 3 independent. ( B ) Caspase-1 in supernatants (SN) of THP-1 cells pretreated with 1.6 mM CaCl 2 and activated by nigericin as indicated and assessed by ELISA. Data are means ± SEM of at least 3 independent. ( C ) Actin depolymerization was performed with 0.2 μg/ml of pyrene-labeled F-actin containing nigericin-activated THP-1 cells lysate. Representative pictures of 3 independent experiments. ( D ) Area under the curve (AUC) represented in ( C ) was calculated using GraphPad Prism version 6. Data are represented as mean ± SEM of at least 3 independent experiments. Statistical significance was determined by Mann-Whitney U analysis. See Figure S4 .

    Article Snippet: Culture supernatants were tested for the production of IL-1β or caspase-1 by enzyme immunoassay (Human IL-1β, eBioscience, San Diego, CA, USA and Caspase-1, Quantikine; R & D Systems, Minneapolis, MN, USA).

    Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Labeling, MANN-WHITNEY

    FliI and LRRFIP2 enable inhibition of NLRP3 inflammasome activity via co-localization with F-actin. ( A ) Confocal microscopy of ATP- and nigericin-activated THP-1 cells. Blue, nuclei. Outlined areas are enlarged in top right corners. Scale bars, 10 μm. Representative pictures of 3 independent experiments. ( B ) Western blot analysis of FliI and LRRFIP2 expression in THP-1 cells stably transduced with lentivirus carrying FliI and LRRFIP2 shRNA. Data are representative of 3 independent experiments. ( C ) Confocal microscopy of ATP- and nigericin-activated THP-1 cells transduced with FliI and LRRFIP2 shRNA. Blue, nuclei. Outlined areas are enlarged in top right corners. Scale bars, 10 μm. Representative pictures of 3 independent experiments. ( D ) IL-1β production in culture supernatants of THP-1 cells transduced with FliI and LRRFIP2 shRNA and unstimulated or stimulated with ATP and nigericin. Data are means ± SEM of at least 3 independent. ( E ) Caspase-1 in supernatants (SN) of THP-1 cells treated as indicated and assessed by ELISA (upper panels) and Western blot (bottom panels); the presence of caspase-1 was assessed by Western blot into cells lysate. Data are represented as mean ± SEM of at least 3 independent experiments or representative of 3 independent experiments. Statistical significance was determined by Mann-Whitney U analysis. The cropped blots were run under the same experimental conditions. See Figure S3 .

    Journal: Scientific Reports

    Article Title: F-actin dampens NLRP3 inflammasome activity via Flightless-I and LRRFIP2

    doi: 10.1038/srep29834

    Figure Lengend Snippet: FliI and LRRFIP2 enable inhibition of NLRP3 inflammasome activity via co-localization with F-actin. ( A ) Confocal microscopy of ATP- and nigericin-activated THP-1 cells. Blue, nuclei. Outlined areas are enlarged in top right corners. Scale bars, 10 μm. Representative pictures of 3 independent experiments. ( B ) Western blot analysis of FliI and LRRFIP2 expression in THP-1 cells stably transduced with lentivirus carrying FliI and LRRFIP2 shRNA. Data are representative of 3 independent experiments. ( C ) Confocal microscopy of ATP- and nigericin-activated THP-1 cells transduced with FliI and LRRFIP2 shRNA. Blue, nuclei. Outlined areas are enlarged in top right corners. Scale bars, 10 μm. Representative pictures of 3 independent experiments. ( D ) IL-1β production in culture supernatants of THP-1 cells transduced with FliI and LRRFIP2 shRNA and unstimulated or stimulated with ATP and nigericin. Data are means ± SEM of at least 3 independent. ( E ) Caspase-1 in supernatants (SN) of THP-1 cells treated as indicated and assessed by ELISA (upper panels) and Western blot (bottom panels); the presence of caspase-1 was assessed by Western blot into cells lysate. Data are represented as mean ± SEM of at least 3 independent experiments or representative of 3 independent experiments. Statistical significance was determined by Mann-Whitney U analysis. The cropped blots were run under the same experimental conditions. See Figure S3 .

    Article Snippet: Culture supernatants were tested for the production of IL-1β or caspase-1 by enzyme immunoassay (Human IL-1β, eBioscience, San Diego, CA, USA and Caspase-1, Quantikine; R & D Systems, Minneapolis, MN, USA).

    Techniques: Inhibition, Activity Assay, Confocal Microscopy, Western Blot, Expressing, Stable Transfection, Transduction, shRNA, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY