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  • 98
    Thermo Fisher nigericin
    Nigericin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 98/100, based on 322 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore nigericin
    Pellino2 ubiquitinates IRAK1 and suppress the inhibitory effects of IRAK1 on NLRP3. a Immunoblot analysis of myc and IRAK1 in lysates (Input) and immunoprecipitated (IP) myc samples from HEK293T cells transfected with myc-tagged Pellino2 and untagged IRAK1. b Immunoblot analysis of HA, IRAK1 and FLAG in lysates (Input) and IP IRAK1 samples from HEK293T cells transfected with FLAG-tagged Pellino2, FLAG-tagged Pellino2 RING mutant, untagged IRAK1, and HA-Ubiquitin. c, d Immunoblot analysis of Ubiquitin (short exposure (SE) or long exposure (LE)) and IRAK1 in lysates (Input) and IP IRAK1 samples or IRAK1 and ubq in TUBE-Ubq elution and cell lysates ( d ) from WT and Peli2 −/− BMDMs treated with 100 ng/ml of LPS for the indicated times. e Immunoblot analysis of NLRP3 and IRAK1 in lysates (Input) and IP IRAK1 samples from WT and Peli2 −/− BMDMs treated with 100 ng/ml LPS for the indicated times. f ELISA of IL-1β (left panel) and TNF (right panel) in medium from primary WT and Irak1 −/− BMDMs treated with 100 ng/ml LPS for 3 h and then with 2.5 mM ATP or 5 mM <t>Nigericin</t> for 1 h. UT, untreated. g Immunoblot analysis of IL-1β and Caspase-1 in medium (Sup) and lysates of WT and Irak1 −/− BMDMs stimulated with 100 ng/ml LPS for 3 h and 2.5 mM ATP for 1 h. h Immunoblot analysis of HA, NLRP3, and IRAK1 in lysates (Input) and IP NLRP3 samples from HEK293T cells transfected with V5-tagged NLRP3, untagged IRAK1, untagged kinase dead IRAK1 (IRAK1-KD), and HA-ubiquitin. β-Actin was used as loading controls. i Immunoblot analysis of Ubiquitin, NLRP3, and IRAK1 in lysates (Input) and IP NLRP3 samples from immortalized WT and Irak1 −/− BMDMs treated with 100 ng/ml LPS for the indicated times. j Immortalized WT and Irak1 −/− BMDMs were infected with MSCV as control (Ctrl) or with MSCV containing an expression construct encoding myc-tagged murine Pellino2 (Peli2), Pellino2 RING mutant (RING) or Pellino2 FHA mutant (FHA). Immunoblot analysis of myc and NLRP3 in lysates (Input) and IP myc samples from virus-infected BMDMs treated with 100 ng/ml LPS for indicated times. * p
    Nigericin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2148 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    InvivoGen nigericin
    Effects of POH1 expression on inflammasome-mediated ASC foci formation. a-c Poh1 Δ/+ and Poh1 Δ/Δ BMDMs were primed with LPS for 12–14 h and then stimulated with ATP (0.5 h), <t>nigericin</t> (Nig, 0.5 h), poly(dA:dT) (5 h) or flagellin (5 h) as indicated, a cells were then fixed and stained for ASC (green) and DNA (blue) (scale bars, 10 µm); b percentages of macrophages containing ASC foci were calculated; or c cells harvested after different treatments were analysed for ASC polymerization. d , e Immunofluorescence microscopy of BMDMs transduced with control (Vector) or POH1, the cells were treated as in a , d and then stained for ASC (green) and DNA (blue) (scale bars, 10 µm); e percentages of macrophages containing ASC foci. f Immunoblot analysis of ASC polymerization in BMDMs treated as in d . g , h HEK293T cells were transfected with 200 ng of plasmids encoding Flag-pro-caspase-1, Flag-ASC and His-pro-IL-1β with different amounts (31.25, 62.5, 125, 250, 500 ng) of plasmid encoding Flag-POH1 and 500 ng of control plasmid, g the cell lysates were immunoblotted with the indicated antibodies; h IL-1β levels in supernatants were analysed by ELISA 36 h after transfection. Similar results were obtained from three independent experiments. The results represent the mean ± s.d. of three independent sets of experiments. *** p
    Nigericin, supplied by InvivoGen, used in various techniques. Bioz Stars score: 99/100, based on 360 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Tocris nigericin
    Effects of heavy metals on NLRP3 inflammasome activation. ( A ) Schematic diagram for inflammasome activation. BMDMs (1 × 10 6 cells per well) were treated with LPS (1 μg/ml) as a 1 st signal for priming, after which cells were replaced with media containing an inflammasome trigger (2 nd signal) with/without heavy metals (HMs) as indicated. Inflammasome activation was assessed by ELISA and/or immunoblotting. ( B ) LPS-primed BMDMs were treated with <t>nigericin</t> (NG) in the presence of media only (Non), cadmium (Cd), mercury (Hg), arsenic (As), or lead (Pb). Release of IL-1β was analyzed by ELISA. ( C ) LPS-primed BMDMs were subjected to activation of NLRP3 inflammasome by NG, monosodium urate crystal (MSU), or aluminum (Alum) treatment in the presence of an increasing dosage of mercury (Hg). The cleaved form of caspase-1 (Casp1, p20) was detected by immunoblotting, and secretion of IL-1β was observed with ELISA. ( D ) LPS-primed BMDMs were treated with NG, MSU, or Alum in the presence of arsenic (As). Release of Casp1 and IL-1β was analyzed with immunoblotting and ELISA. All immunoblot data shown are representative of at least three independent experiments. Bar graph presents the mean ± SD with at least two independent experiments.
    Nigericin, supplied by Tocris, used in various techniques. Bioz Stars score: 94/100, based on 59 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Cayman Chemical nigericin
    Inhibition of MIF prevents NLRP3 inflammasome activation. ASC-cerulean macrophages were primed with LPS (10 ng ml − 1 ) overnight. The following day cells were treated with COR123625 (50 µM) for 2 h before activation of the inflammasome with a <t>nigericin</t> (10 µM) (1 h) or b silica (150 µg ml − 1 ) (4 h). Confocal images are representative of three independent experiments. c , d Data are presented as the percentage of ASC-speck-positive cells. Data shown are mean ± SEM of three independent experiments. e WT BMDMs were left untreated, treated with LPS alone (100 ng ml − 1 ) for 5 h, treated with COR123625 for 1 h prior to the addition of LPS, primed with LPS before inflammasome activation with nigericin (5 µM) for 1 h, or treated with COR123625 before LPS and nigericin stimulation. Levels of LDH release were quantified using the Promega cytotoxicity assay. f WT or Mif − / − BMDMs were treated with LPS (10 ng ml − 1 ) + nigericin (5 μM) and lysates and supernatants analyzed by Western blot for caspase-1 and IL-1β. Images are representative of > 3 mice. g BMDMs from WT and Mif − / − mice were treated with (10 ng ml − 1 ) + nigericin (5 μM), fixed and stained for ASC, and analyzed by confocal microscopy. Images are z projections of multiple z-stacks. h Quantitation of ASC specks in g , n = 3 mice per group. Data are expressed as percentage increase of mean ± SEM from three mice. * P
    Nigericin, supplied by Cayman Chemical, used in various techniques. Bioz Stars score: 92/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Enzo Biochem nigericin
    MARK4 interacts with NLRP3 in a microtubule-dependent manner. ( a ) Upon <t>nigericin</t> stimulation (3 μM for 2 h), microtubule-disrupting drugs colchicine and nocodazole reduced the interaction between Mark4 and Nlrp3, shown by PLA signals in WT BMDM cells. Mark4 KO and Nlrp3 KO BMDM cells were employed as controls. ( b ) MARK4 was associated with NLRP3 in co-immunoprecipitation assay. Whole cell lysates were analysed as indication of transfection. Western blots are representative of 3 independent experiments. ( c ) Upon nigericin stimulation, PLA signal of NlrpP3 and Mark4, or Asc and Mark4 in BMDM cells derived from WT or Nlrp3 KO. Secondary only was employed as control in this PLA assay. ( d ) PLA signal of Nlrp3 and Asc in BMDM cells derived from WT or Nlrp3 KO or Asc KO before or after nigericin stimulation (3 μM for 2 h). Mean±s.e.m. for all the cells taken from 5 to 8 different views at × 40 magnification for each group ( a , b , d ). Comparisons of the two different groups were analysed by unpaired t -test. NS was considered as not statistically significant. * P
    Nigericin, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 92/100, based on 51 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Merck KGaA nigericin
    ROS is involved in inflammasome activation induced by ER stress. a ROS production detected by flow cytometry in LPS, TG, or LPS plus TG-treated BMDMs (UT unstimulated). b ROS production in LPS plus TG-treated BMDMs with or without Nec-1 pretreatment. c ROS production in LPS plus TG-treated BMDMs transfected with control siRNA (Scr) or RIP1-specific siRNAs. d ROS production in LPS plus TG-treated BMDMs with or without Midivi-1 or BHA pretreatment. e , f Secretion of IL-1β and TNF-α by BMDMs stimulated by LPS plus TG or LPS plus <t>Nigericin</t> with or without antioxidant BHA pretreatment. g Immunoblot of cleaved caspase-1 (p20) in cell supernatants (SN) and caspase-1 (pro caspase-1) and β-actin in cell lysates (Input) of BMDMs stimulated with LPS plus TG or LPS plus Nigericin with or without BHA pretreatment. Figures are representative of at least three independent experiments. Bars indicate means plus SD. ns not significant, ** P
    Nigericin, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 92/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore ionophore nigericin
    ROS is involved in inflammasome activation induced by ER stress. a ROS production detected by flow cytometry in LPS, TG, or LPS plus TG-treated BMDMs (UT unstimulated). b ROS production in LPS plus TG-treated BMDMs with or without Nec-1 pretreatment. c ROS production in LPS plus TG-treated BMDMs transfected with control siRNA (Scr) or RIP1-specific siRNAs. d ROS production in LPS plus TG-treated BMDMs with or without Midivi-1 or BHA pretreatment. e , f Secretion of IL-1β and TNF-α by BMDMs stimulated by LPS plus TG or LPS plus <t>Nigericin</t> with or without antioxidant BHA pretreatment. g Immunoblot of cleaved caspase-1 (p20) in cell supernatants (SN) and caspase-1 (pro caspase-1) and β-actin in cell lysates (Input) of BMDMs stimulated with LPS plus TG or LPS plus Nigericin with or without BHA pretreatment. Figures are representative of at least three independent experiments. Bars indicate means plus SD. ns not significant, ** P
    Ionophore Nigericin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 31 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    FUJIFILM nigericin
    ROS is involved in inflammasome activation induced by ER stress. a ROS production detected by flow cytometry in LPS, TG, or LPS plus TG-treated BMDMs (UT unstimulated). b ROS production in LPS plus TG-treated BMDMs with or without Nec-1 pretreatment. c ROS production in LPS plus TG-treated BMDMs transfected with control siRNA (Scr) or RIP1-specific siRNAs. d ROS production in LPS plus TG-treated BMDMs with or without Midivi-1 or BHA pretreatment. e , f Secretion of IL-1β and TNF-α by BMDMs stimulated by LPS plus TG or LPS plus <t>Nigericin</t> with or without antioxidant BHA pretreatment. g Immunoblot of cleaved caspase-1 (p20) in cell supernatants (SN) and caspase-1 (pro caspase-1) and β-actin in cell lysates (Input) of BMDMs stimulated with LPS plus TG or LPS plus Nigericin with or without BHA pretreatment. Figures are representative of at least three independent experiments. Bars indicate means plus SD. ns not significant, ** P
    Nigericin, supplied by FUJIFILM, used in various techniques. Bioz Stars score: 92/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Adipogen nigericin
    Extracellular specks represent a cell-derived danger signal. ( a ) Confocal imaging of iMøs incubated with in vitro -assembled ASC-mCerulean specks for 2 hours. Scale bar 9 μm. ( b ) Immunoblot of ASC-mCerulean (anti-GFP) in lysates of BMDMs incubated with ASC-mCerulean specks. Non-phagocyted specks were washed away. ( c ) Confocal imaging of iMøs incubated with Dextran-A488 (20 μg/ml) alone or together with the lysosomal damaging LeuLeu-O-Me (0.5 μM), or ASC-mCerulean specks (100 μg/ml) for 4 hours. Scale bars: 7.0 μm top left, 4.9 μm top right, 6.0 μm bottom left, and 2.9 μm bottom right. ( d ) Average size of lysosomes in cells treated as indicated. ( e ) Percentage of recipient ASC-mCherry cells containing ASC-mCherry specks after incubation with silica crystals (100 μg/ml), <t>nigericin</t> (10 μM) or ASC-mCerulean specks (100 μg/ml). ( f-g ) ELISA of IL-1β in supernatants of LPS-primed (200 ng/ml, 2 h) BMDMs that were either left untreated or stimulated with cholesterol crystals (250 μg/ml), ASC specks, or a mock preparation from Pycard −/− iMøs (200 μg/ml). Data are representative from three independent experiments ( a - e ); d , mean + SD of combined data from two independent experiments, in which at least 5 fields/condition were analyzed (Mann-Whitney test, **P
    Nigericin, supplied by Adipogen, used in various techniques. Bioz Stars score: 92/100, based on 65 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Enzo Biochem nigericin sodium salt
    Trim21 exposes AdV genomes to cGAS and STING to trigger NLRP3‐dependent inflammasome activation AIM2 mRNA levels from PBMCs, CD19 +ve B cells, CD14 +ve monocytes or HMDM derived from CD14 +ve monocytes were assessed by qPCR, and copy number was determined relative to actin copy number ( n = 5 mean ± s.e.m). HMDM were stimulated with AdV (50,000 pp/cell) and 20 μg/ml h9C12 for 1 h, washed 2× with SFM and then whole cell lysates harvested after a further 5 h. Viral hexon and transgene (GFP) expression in the cytosol was assessed by Western blot. Data are representative of two independent experiments. THP‐1s expressing either a control guide RNA or targeting cGAS and STING were generated and stimulated with 1,000 U/ml IFN‐α for 4 h and protein levels assessed by Western blot. THP‐1s deficient in cGAS and STING were stimulated with AdV (50,000 pp/cell) and 20 μg/ml h9C12, 200 ng/well HT‐DNA, 10 μM <t>Nigericin</t> or 10 ng/ml LPS for 16 h. Data show combined data (mean ± s.e.m) of three experiments with absolute protein values (upper panel) or as % cytokine output of KO cells relative to Ctrl‐treated cells (lower panel), * P ≤ 0.05, *** P ≤ 0.001 unpaired, two‐tailed t ‐test). WT THP‐1s were treated with 5 μM H151 for 30 min before stimulation as in (D). Data in upper panel are representative of three independent experiments. Data in lower panel show combined data of these three experiments (mean ± s.e.m) showing H151‐treated cells relative to media treated cells (* P ≤ 0.05, *** P ≤ 0.001 unpaired, two‐tailed t ‐test). ASC‐GFP THP‐1s were treated with 5 μM H151 for 30 min before stimulation with AdV‐mCherry (50,000 pp/cell) + 20 μg/ml h9C12 or 20 mg/ml IVIg or 200 ng/ well HT‐DNA for 8 h. A representative image (scale bar 50 μm) and quantification of number of cells with ASC specks from one representative experiment of three are shown. Source data are available online for this figure.
    Nigericin Sodium Salt, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 92/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Santa Cruz Biotechnology nigericin
    Inhibition of AR prevented –HG-induced NLRP3 inflammasome–mediated release of innate immune cytokines in Thp1 cells. Thp1 cells were preincubated (4 hours) with HG (25 mM) and then incubated with fidarestat (10 μΜ) for 30 minutes, followed by ATP (2 mM) for 1 hour. (a) Equal amount of proteins in the cell extracts (CEs) were subjected to Western blot analysis to determine the expression of inflammasome complex proteins NLRP3, ASC, caspase-1, and innate immune cytokines IL-1 β and IL-18, using specific antibodies. Equal amounts of proteins in the lyophilized culture media (CM) were also subjected to Western blot analysis for IL-1 β and IL-18. (b) IL-1 β release in cell culture supernatant was also determined by a specific ELISA kit. (c) IL-1 β mRNA levels in Thp1 cells were determined by reverse transcription-PCR, as described in Methods. (d) Thp1 cells were incubated (4 hours) with HG (25 mM) and then incubated with fidarestat (10 μM) for 30 minutes, followed by ATP (2 mM) and <t>nigericin</t> (20 µM) for 1 hour without or with addition of KCl (130 mM). Equal amounts of proteins in the lyophilized CM andCEs were subjected to Western blot analysis for IL-1 β . The representative cropped blots are shown. The bars represent the mean ± standard error of the mean (n ≥ 4). Fid, fidarestat.
    Nigericin, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    AG Scientific nigericin
    Inhibition of AR prevented –HG-induced NLRP3 inflammasome–mediated release of innate immune cytokines in Thp1 cells. Thp1 cells were preincubated (4 hours) with HG (25 mM) and then incubated with fidarestat (10 μΜ) for 30 minutes, followed by ATP (2 mM) for 1 hour. (a) Equal amount of proteins in the cell extracts (CEs) were subjected to Western blot analysis to determine the expression of inflammasome complex proteins NLRP3, ASC, caspase-1, and innate immune cytokines IL-1 β and IL-18, using specific antibodies. Equal amounts of proteins in the lyophilized culture media (CM) were also subjected to Western blot analysis for IL-1 β and IL-18. (b) IL-1 β release in cell culture supernatant was also determined by a specific ELISA kit. (c) IL-1 β mRNA levels in Thp1 cells were determined by reverse transcription-PCR, as described in Methods. (d) Thp1 cells were incubated (4 hours) with HG (25 mM) and then incubated with fidarestat (10 μM) for 30 minutes, followed by ATP (2 mM) and <t>nigericin</t> (20 µM) for 1 hour without or with addition of KCl (130 mM). Equal amounts of proteins in the lyophilized CM andCEs were subjected to Western blot analysis for IL-1 β . The representative cropped blots are shown. The bars represent the mean ± standard error of the mean (n ≥ 4). Fid, fidarestat.
    Nigericin, supplied by AG Scientific, used in various techniques. Bioz Stars score: 91/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Applichem nigericin
    Febuxostat restored intracellular ATP by activating salvage pathway. ( a ) Schema of purine metabolism. ( b ) Primed BMDMs were pretreated 30 min with vehicle or febuxostat, and then stimulated 90 min with <t>nigericin.</t> Intracellular ATP, ADP and AMP were measured by HPLC method. Total adenylate was calculated. Intracellular uric acid level was measured. Data are representative of two independent experiments in which the same data were obtained and shown as mean ± SD. * p
    Nigericin, supplied by Applichem, used in various techniques. Bioz Stars score: 90/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Adipogen m nigericin
    A model integrating planarian bioelectrics to regenerative outcomes. ( A ) Cells at the surfaces of wound blastema (inserts) are predicted to measure the difference between their own depolarization (V mem (B)) and the average depolarization (V mem (Int)) of neighboring cells just interior to the wound blastema. If this difference is larger than some threshold value, the brain-head pathway is activated; if the difference is smaller than this threshold or negative, the tail pathway is activated. Branching between pathways is modeled by logistic-function kinetics. Local mutual inhibition by Notum and β -catenin is active in otherwise-untreated WT animals. ( B ) Treatment with <t>nigericin</t> solution (symbolized by orange lightning bolt arrow ) immediately after amputation depolarizes both wound blastema, leading to brain-head pathway activation and head regeneration at both wounds. Excessive blastema depolarization turns local mutual inhibition by Notum and β -catenin off. To see this figure in color, go online.
    M Nigericin, supplied by Adipogen, used in various techniques. Bioz Stars score: 93/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Pellino2 ubiquitinates IRAK1 and suppress the inhibitory effects of IRAK1 on NLRP3. a Immunoblot analysis of myc and IRAK1 in lysates (Input) and immunoprecipitated (IP) myc samples from HEK293T cells transfected with myc-tagged Pellino2 and untagged IRAK1. b Immunoblot analysis of HA, IRAK1 and FLAG in lysates (Input) and IP IRAK1 samples from HEK293T cells transfected with FLAG-tagged Pellino2, FLAG-tagged Pellino2 RING mutant, untagged IRAK1, and HA-Ubiquitin. c, d Immunoblot analysis of Ubiquitin (short exposure (SE) or long exposure (LE)) and IRAK1 in lysates (Input) and IP IRAK1 samples or IRAK1 and ubq in TUBE-Ubq elution and cell lysates ( d ) from WT and Peli2 −/− BMDMs treated with 100 ng/ml of LPS for the indicated times. e Immunoblot analysis of NLRP3 and IRAK1 in lysates (Input) and IP IRAK1 samples from WT and Peli2 −/− BMDMs treated with 100 ng/ml LPS for the indicated times. f ELISA of IL-1β (left panel) and TNF (right panel) in medium from primary WT and Irak1 −/− BMDMs treated with 100 ng/ml LPS for 3 h and then with 2.5 mM ATP or 5 mM Nigericin for 1 h. UT, untreated. g Immunoblot analysis of IL-1β and Caspase-1 in medium (Sup) and lysates of WT and Irak1 −/− BMDMs stimulated with 100 ng/ml LPS for 3 h and 2.5 mM ATP for 1 h. h Immunoblot analysis of HA, NLRP3, and IRAK1 in lysates (Input) and IP NLRP3 samples from HEK293T cells transfected with V5-tagged NLRP3, untagged IRAK1, untagged kinase dead IRAK1 (IRAK1-KD), and HA-ubiquitin. β-Actin was used as loading controls. i Immunoblot analysis of Ubiquitin, NLRP3, and IRAK1 in lysates (Input) and IP NLRP3 samples from immortalized WT and Irak1 −/− BMDMs treated with 100 ng/ml LPS for the indicated times. j Immortalized WT and Irak1 −/− BMDMs were infected with MSCV as control (Ctrl) or with MSCV containing an expression construct encoding myc-tagged murine Pellino2 (Peli2), Pellino2 RING mutant (RING) or Pellino2 FHA mutant (FHA). Immunoblot analysis of myc and NLRP3 in lysates (Input) and IP myc samples from virus-infected BMDMs treated with 100 ng/ml LPS for indicated times. * p

    Journal: Nature Communications

    Article Title: The E3 ubiquitin ligase Pellino2 mediates priming of the NLRP3 inflammasome

    doi: 10.1038/s41467-018-03669-z

    Figure Lengend Snippet: Pellino2 ubiquitinates IRAK1 and suppress the inhibitory effects of IRAK1 on NLRP3. a Immunoblot analysis of myc and IRAK1 in lysates (Input) and immunoprecipitated (IP) myc samples from HEK293T cells transfected with myc-tagged Pellino2 and untagged IRAK1. b Immunoblot analysis of HA, IRAK1 and FLAG in lysates (Input) and IP IRAK1 samples from HEK293T cells transfected with FLAG-tagged Pellino2, FLAG-tagged Pellino2 RING mutant, untagged IRAK1, and HA-Ubiquitin. c, d Immunoblot analysis of Ubiquitin (short exposure (SE) or long exposure (LE)) and IRAK1 in lysates (Input) and IP IRAK1 samples or IRAK1 and ubq in TUBE-Ubq elution and cell lysates ( d ) from WT and Peli2 −/− BMDMs treated with 100 ng/ml of LPS for the indicated times. e Immunoblot analysis of NLRP3 and IRAK1 in lysates (Input) and IP IRAK1 samples from WT and Peli2 −/− BMDMs treated with 100 ng/ml LPS for the indicated times. f ELISA of IL-1β (left panel) and TNF (right panel) in medium from primary WT and Irak1 −/− BMDMs treated with 100 ng/ml LPS for 3 h and then with 2.5 mM ATP or 5 mM Nigericin for 1 h. UT, untreated. g Immunoblot analysis of IL-1β and Caspase-1 in medium (Sup) and lysates of WT and Irak1 −/− BMDMs stimulated with 100 ng/ml LPS for 3 h and 2.5 mM ATP for 1 h. h Immunoblot analysis of HA, NLRP3, and IRAK1 in lysates (Input) and IP NLRP3 samples from HEK293T cells transfected with V5-tagged NLRP3, untagged IRAK1, untagged kinase dead IRAK1 (IRAK1-KD), and HA-ubiquitin. β-Actin was used as loading controls. i Immunoblot analysis of Ubiquitin, NLRP3, and IRAK1 in lysates (Input) and IP NLRP3 samples from immortalized WT and Irak1 −/− BMDMs treated with 100 ng/ml LPS for the indicated times. j Immortalized WT and Irak1 −/− BMDMs were infected with MSCV as control (Ctrl) or with MSCV containing an expression construct encoding myc-tagged murine Pellino2 (Peli2), Pellino2 RING mutant (RING) or Pellino2 FHA mutant (FHA). Immunoblot analysis of myc and NLRP3 in lysates (Input) and IP myc samples from virus-infected BMDMs treated with 100 ng/ml LPS for indicated times. * p

    Article Snippet: Adenosine 5′-triphosphate disodium salt hydrate (ATP) (A1852) and Nigericin (N7143) were from Sigma.

    Techniques: Immunoprecipitation, Transfection, Mutagenesis, Enzyme-linked Immunosorbent Assay, Infection, Expressing, Construct

    Pellino2 mediates NLRP3-dependent oligomerization of ASC. a Immunofluorescence staining of ASC in WT and Peli2 −/− BMDMs that were left untreated (UT) or treated with 100 ng/ml LPS for 3 h and further stimulated with ATP for 30 min. ASC specks were detected by immunostaining using anti-ASC antibody and anti-rabbit Alexa Fluor 568 (red) and cells were counter stained with nuclei-staining DAPI. The histogram quantitates the percentage of cells that exhibit ASC speck formation. (scale bar = 100 μm). b, c Immunoblot analysis of ASC in chemically cross-linked NP-40 insoluble fractions and in NP-40 soluble fractions from cell lysates of WT and Peli2 −/− BMDMs stimulated with 100 ng/ml LPS for 3 h with or without further treatment with b 2.5 mM ATP, or c 5 mM Nigericin for 30 min. β-Actin was used as loading controls. d WT and Peli2 −/− BMDMs were infected with MSCV as control (Ctrl) or with MSCV containing an expression construct encoding myc-tagged murine Pellino2 (Peli2), Pellino2 RING mutant (RING), or Pellino2 FHA mutant (FHA). Immunoblot analysis of ASC in chemically cross-linked NP-40 insoluble fractions and in NP-40 soluble fractions from cell lysates of MSCV-infected cells treated with 100 ng/ml LPS for 3 h followed by 2.5 mM ATP for 30 min. The expression of the Pellino2 constructs was measured by immunoblotting with an anti-myc antibody. ** p

    Journal: Nature Communications

    Article Title: The E3 ubiquitin ligase Pellino2 mediates priming of the NLRP3 inflammasome

    doi: 10.1038/s41467-018-03669-z

    Figure Lengend Snippet: Pellino2 mediates NLRP3-dependent oligomerization of ASC. a Immunofluorescence staining of ASC in WT and Peli2 −/− BMDMs that were left untreated (UT) or treated with 100 ng/ml LPS for 3 h and further stimulated with ATP for 30 min. ASC specks were detected by immunostaining using anti-ASC antibody and anti-rabbit Alexa Fluor 568 (red) and cells were counter stained with nuclei-staining DAPI. The histogram quantitates the percentage of cells that exhibit ASC speck formation. (scale bar = 100 μm). b, c Immunoblot analysis of ASC in chemically cross-linked NP-40 insoluble fractions and in NP-40 soluble fractions from cell lysates of WT and Peli2 −/− BMDMs stimulated with 100 ng/ml LPS for 3 h with or without further treatment with b 2.5 mM ATP, or c 5 mM Nigericin for 30 min. β-Actin was used as loading controls. d WT and Peli2 −/− BMDMs were infected with MSCV as control (Ctrl) or with MSCV containing an expression construct encoding myc-tagged murine Pellino2 (Peli2), Pellino2 RING mutant (RING), or Pellino2 FHA mutant (FHA). Immunoblot analysis of ASC in chemically cross-linked NP-40 insoluble fractions and in NP-40 soluble fractions from cell lysates of MSCV-infected cells treated with 100 ng/ml LPS for 3 h followed by 2.5 mM ATP for 30 min. The expression of the Pellino2 constructs was measured by immunoblotting with an anti-myc antibody. ** p

    Article Snippet: Adenosine 5′-triphosphate disodium salt hydrate (ATP) (A1852) and Nigericin (N7143) were from Sigma.

    Techniques: Immunofluorescence, Staining, Immunostaining, Infection, Expressing, Construct, Mutagenesis

    Pellino2 mediates activation of the NLRP3 pathway. ELISA of a IL-1β and b IL-18, and c LDH assay of medium from WT and Peli2 −/− BMDMs treated with 100 ng/ml LPS for 3 h with or without further stimulation with 2.5 mM ATP for 1 h. UT, untreated. d Immunoblot analysis of IL-1β and Caspase-1 in medium (Sup) and lysates from WT and Peli2 −/− BMDMs stimulated with 100 ng/ml LPS for 3 h with or without further stimulation with 2.5 mM ATP for 1 h or transfection of Poly (dA:dT) (1 μg/ml) for 6 h. ELISA of e IL-1β and f IL-18 and g LDH assay of medium from WT and Peli2 −/− BMDMs treated with 100 ng/ml LPS for 3 h with or without further stimulation with 5 mM Nigericin for 1 h. h Immunoblot analysis of IL-1β and Caspase-1 in medium (Sup) and lysates from WT and Peli2 −/− BMDMs stimulated with 100 ng/ml LPS for 3 h with or without further stimulation with 5 mM Nigericin for 1 h or transfection of Poly (dA:dT) (1 μg/ml) for 6 h. i ELISA of IL-1β of medium from WT and Peli2 −/− BMDMs treated with 100 ng/ml LPS for 3 h with or without further stimulation with the indicated concentrations of Alum for 6 h. j ELISA of IL-1β in medium from peritoneal-resident macrophages isolated from WT and Peli2 −/− mice. Cells were treated with 100 ng/ml of LPS for 3 h with or without further stimulation with 2.5 mM ATP for 1 h. k-m Human THP1 cells were transfected with human Pellino2-specific siRNA or control siRNA. k Quantitative RT-PCR analysis of PELI2 expression in transfected cells. l ELISA of IL-1β in medium from transfected THP1 cells stimulated with 100 ng/ml LPS for 6 h with or without further treatment with 2.5 mM ATP or 5 mM Nigericin for 1 h. m Immunoblot analysis of IL-1β and Caspase-1 in medium (Sup) and lysates from transfected THP1 cells stimulated with 100 ng/ml LPS for 6 h with or without further stimulation with 2.5 mM ATP or 5 mM Nigericin for 1 h. β-Actin was used as loading controls. * p

    Journal: Nature Communications

    Article Title: The E3 ubiquitin ligase Pellino2 mediates priming of the NLRP3 inflammasome

    doi: 10.1038/s41467-018-03669-z

    Figure Lengend Snippet: Pellino2 mediates activation of the NLRP3 pathway. ELISA of a IL-1β and b IL-18, and c LDH assay of medium from WT and Peli2 −/− BMDMs treated with 100 ng/ml LPS for 3 h with or without further stimulation with 2.5 mM ATP for 1 h. UT, untreated. d Immunoblot analysis of IL-1β and Caspase-1 in medium (Sup) and lysates from WT and Peli2 −/− BMDMs stimulated with 100 ng/ml LPS for 3 h with or without further stimulation with 2.5 mM ATP for 1 h or transfection of Poly (dA:dT) (1 μg/ml) for 6 h. ELISA of e IL-1β and f IL-18 and g LDH assay of medium from WT and Peli2 −/− BMDMs treated with 100 ng/ml LPS for 3 h with or without further stimulation with 5 mM Nigericin for 1 h. h Immunoblot analysis of IL-1β and Caspase-1 in medium (Sup) and lysates from WT and Peli2 −/− BMDMs stimulated with 100 ng/ml LPS for 3 h with or without further stimulation with 5 mM Nigericin for 1 h or transfection of Poly (dA:dT) (1 μg/ml) for 6 h. i ELISA of IL-1β of medium from WT and Peli2 −/− BMDMs treated with 100 ng/ml LPS for 3 h with or without further stimulation with the indicated concentrations of Alum for 6 h. j ELISA of IL-1β in medium from peritoneal-resident macrophages isolated from WT and Peli2 −/− mice. Cells were treated with 100 ng/ml of LPS for 3 h with or without further stimulation with 2.5 mM ATP for 1 h. k-m Human THP1 cells were transfected with human Pellino2-specific siRNA or control siRNA. k Quantitative RT-PCR analysis of PELI2 expression in transfected cells. l ELISA of IL-1β in medium from transfected THP1 cells stimulated with 100 ng/ml LPS for 6 h with or without further treatment with 2.5 mM ATP or 5 mM Nigericin for 1 h. m Immunoblot analysis of IL-1β and Caspase-1 in medium (Sup) and lysates from transfected THP1 cells stimulated with 100 ng/ml LPS for 6 h with or without further stimulation with 2.5 mM ATP or 5 mM Nigericin for 1 h. β-Actin was used as loading controls. * p

    Article Snippet: Adenosine 5′-triphosphate disodium salt hydrate (ATP) (A1852) and Nigericin (N7143) were from Sigma.

    Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Lactate Dehydrogenase Assay, Transfection, Isolation, Mouse Assay, Quantitative RT-PCR, Expressing

    Caspase-11 requires IDL but not CDL processing for inducing cell death and IL-1β release. Caspase-11 WT, catalytic mutant (C254A), CDL mutant (CDL uncl ), or IDL mutant (IDL uncl ) were retrovirally expressed in Casp11 −/− BMM. Cells were primed for 12 h with Pam 3 CSK 4 or 4 h with LPS, and transfected with ultrapure K12 E. coli LPS 10 μg/ml for 6 h or exposed to 5 μM nigericin for 2 h. (A) Western blot assessed expression of caspase-11 mutants in cell extracts of Pam 3 CSK 4 -primed, untransfected BMM. (B) Immunoblot detected mature IL-1β and the caspase-11 or caspase-1 large subunits in the culture medium (SUP) and cell extracts (XT) of Pam 3 CSK 4 -primed BMMs transfected with LPS for 6 h. (C) Cell death and (D) IL-1β secretion was assessed 6 h after LPS transfection or 2 h after nigericin exposure. Western blots are representative of three biological replicate experiments. Graphs are mean + SEM of four biological replicate experiments, with significance assessed using a Mann–Whitney test.

    Journal: Life Science Alliance

    Article Title: Dimerization and auto-processing induce caspase-11 protease activation within the non-canonical inflammasome

    doi: 10.26508/lsa.201800237

    Figure Lengend Snippet: Caspase-11 requires IDL but not CDL processing for inducing cell death and IL-1β release. Caspase-11 WT, catalytic mutant (C254A), CDL mutant (CDL uncl ), or IDL mutant (IDL uncl ) were retrovirally expressed in Casp11 −/− BMM. Cells were primed for 12 h with Pam 3 CSK 4 or 4 h with LPS, and transfected with ultrapure K12 E. coli LPS 10 μg/ml for 6 h or exposed to 5 μM nigericin for 2 h. (A) Western blot assessed expression of caspase-11 mutants in cell extracts of Pam 3 CSK 4 -primed, untransfected BMM. (B) Immunoblot detected mature IL-1β and the caspase-11 or caspase-1 large subunits in the culture medium (SUP) and cell extracts (XT) of Pam 3 CSK 4 -primed BMMs transfected with LPS for 6 h. (C) Cell death and (D) IL-1β secretion was assessed 6 h after LPS transfection or 2 h after nigericin exposure. Western blots are representative of three biological replicate experiments. Graphs are mean + SEM of four biological replicate experiments, with significance assessed using a Mann–Whitney test.

    Article Snippet: To activate the NLRP3 inflammasome, BMM were first primed for 4 h with 100 ng/ml K12 ultrapure LPS, before the medium was replaced with CSF-1-replete Opti-MEM containing 5 μM Nigericin Sodium salt (Sigma-Aldrich).

    Techniques: Mutagenesis, Transfection, Western Blot, Expressing, MANN-WHITNEY

    Effects of POH1 expression on inflammasome-mediated ASC foci formation. a-c Poh1 Δ/+ and Poh1 Δ/Δ BMDMs were primed with LPS for 12–14 h and then stimulated with ATP (0.5 h), nigericin (Nig, 0.5 h), poly(dA:dT) (5 h) or flagellin (5 h) as indicated, a cells were then fixed and stained for ASC (green) and DNA (blue) (scale bars, 10 µm); b percentages of macrophages containing ASC foci were calculated; or c cells harvested after different treatments were analysed for ASC polymerization. d , e Immunofluorescence microscopy of BMDMs transduced with control (Vector) or POH1, the cells were treated as in a , d and then stained for ASC (green) and DNA (blue) (scale bars, 10 µm); e percentages of macrophages containing ASC foci. f Immunoblot analysis of ASC polymerization in BMDMs treated as in d . g , h HEK293T cells were transfected with 200 ng of plasmids encoding Flag-pro-caspase-1, Flag-ASC and His-pro-IL-1β with different amounts (31.25, 62.5, 125, 250, 500 ng) of plasmid encoding Flag-POH1 and 500 ng of control plasmid, g the cell lysates were immunoblotted with the indicated antibodies; h IL-1β levels in supernatants were analysed by ELISA 36 h after transfection. Similar results were obtained from three independent experiments. The results represent the mean ± s.d. of three independent sets of experiments. *** p

    Journal: Nature Communications

    Article Title: POH1 deubiquitinates pro-interleukin-1β and restricts inflammasome activity

    doi: 10.1038/s41467-018-06455-z

    Figure Lengend Snippet: Effects of POH1 expression on inflammasome-mediated ASC foci formation. a-c Poh1 Δ/+ and Poh1 Δ/Δ BMDMs were primed with LPS for 12–14 h and then stimulated with ATP (0.5 h), nigericin (Nig, 0.5 h), poly(dA:dT) (5 h) or flagellin (5 h) as indicated, a cells were then fixed and stained for ASC (green) and DNA (blue) (scale bars, 10 µm); b percentages of macrophages containing ASC foci were calculated; or c cells harvested after different treatments were analysed for ASC polymerization. d , e Immunofluorescence microscopy of BMDMs transduced with control (Vector) or POH1, the cells were treated as in a , d and then stained for ASC (green) and DNA (blue) (scale bars, 10 µm); e percentages of macrophages containing ASC foci. f Immunoblot analysis of ASC polymerization in BMDMs treated as in d . g , h HEK293T cells were transfected with 200 ng of plasmids encoding Flag-pro-caspase-1, Flag-ASC and His-pro-IL-1β with different amounts (31.25, 62.5, 125, 250, 500 ng) of plasmid encoding Flag-POH1 and 500 ng of control plasmid, g the cell lysates were immunoblotted with the indicated antibodies; h IL-1β levels in supernatants were analysed by ELISA 36 h after transfection. Similar results were obtained from three independent experiments. The results represent the mean ± s.d. of three independent sets of experiments. *** p

    Article Snippet: Stimulation with 5 mM ATP (Sigma-Aldrich) or 10 µM nigericin (Invivogen) was performed for 0.5 h after LPS priming.

    Techniques: Expressing, Staining, Immunofluorescence, Microscopy, Transduction, Plasmid Preparation, Transfection, Enzyme-linked Immunosorbent Assay

    POH1 inhibits inflammasome-induced pro-IL-1β processing. a-c BMDMs from Poh1 Δ/+ and Poh1 Δ/Δ mice were primed with LPS for 12–14 h and then stimulated with ATP (0.5 h), nigericin (Nig, 0.5 h), poly(dA:dT) (5 h) or flagellin (5 h) as indicated, a IL-1β, b IL-6 and c TNFα levels in supernatants were analysed by ELISA. d-f BMDMs lentivirally transduced with control (Vector) or POH1 were stimulated as in a , d IL-1β, e IL-6 and f TNFα levels in supernatants were analysed by ELISA. g, h BMDMs g derived from Poh1 Δ/+ and Poh1 Δ/Δ mice or h infected by indicated lentiviruses were treated as in a , the cell lysates (CL) and supernatants (SN) were collected and immunoblotted with the indicated antibodies. Similar results were obtained from three independent experiments. The results represent the mean ± s.d. of three independent sets of experiments. ** p

    Journal: Nature Communications

    Article Title: POH1 deubiquitinates pro-interleukin-1β and restricts inflammasome activity

    doi: 10.1038/s41467-018-06455-z

    Figure Lengend Snippet: POH1 inhibits inflammasome-induced pro-IL-1β processing. a-c BMDMs from Poh1 Δ/+ and Poh1 Δ/Δ mice were primed with LPS for 12–14 h and then stimulated with ATP (0.5 h), nigericin (Nig, 0.5 h), poly(dA:dT) (5 h) or flagellin (5 h) as indicated, a IL-1β, b IL-6 and c TNFα levels in supernatants were analysed by ELISA. d-f BMDMs lentivirally transduced with control (Vector) or POH1 were stimulated as in a , d IL-1β, e IL-6 and f TNFα levels in supernatants were analysed by ELISA. g, h BMDMs g derived from Poh1 Δ/+ and Poh1 Δ/Δ mice or h infected by indicated lentiviruses were treated as in a , the cell lysates (CL) and supernatants (SN) were collected and immunoblotted with the indicated antibodies. Similar results were obtained from three independent experiments. The results represent the mean ± s.d. of three independent sets of experiments. ** p

    Article Snippet: Stimulation with 5 mM ATP (Sigma-Aldrich) or 10 µM nigericin (Invivogen) was performed for 0.5 h after LPS priming.

    Techniques: Mouse Assay, Enzyme-linked Immunosorbent Assay, Transduction, Plasmid Preparation, Derivative Assay, Infection

    Effects of heavy metals on NLRP3 inflammasome activation. ( A ) Schematic diagram for inflammasome activation. BMDMs (1 × 10 6 cells per well) were treated with LPS (1 μg/ml) as a 1 st signal for priming, after which cells were replaced with media containing an inflammasome trigger (2 nd signal) with/without heavy metals (HMs) as indicated. Inflammasome activation was assessed by ELISA and/or immunoblotting. ( B ) LPS-primed BMDMs were treated with nigericin (NG) in the presence of media only (Non), cadmium (Cd), mercury (Hg), arsenic (As), or lead (Pb). Release of IL-1β was analyzed by ELISA. ( C ) LPS-primed BMDMs were subjected to activation of NLRP3 inflammasome by NG, monosodium urate crystal (MSU), or aluminum (Alum) treatment in the presence of an increasing dosage of mercury (Hg). The cleaved form of caspase-1 (Casp1, p20) was detected by immunoblotting, and secretion of IL-1β was observed with ELISA. ( D ) LPS-primed BMDMs were treated with NG, MSU, or Alum in the presence of arsenic (As). Release of Casp1 and IL-1β was analyzed with immunoblotting and ELISA. All immunoblot data shown are representative of at least three independent experiments. Bar graph presents the mean ± SD with at least two independent experiments.

    Journal: Scientific Reports

    Article Title: Mercury and arsenic attenuate canonical and non-canonical NLRP3 inflammasome activation

    doi: 10.1038/s41598-018-31717-7

    Figure Lengend Snippet: Effects of heavy metals on NLRP3 inflammasome activation. ( A ) Schematic diagram for inflammasome activation. BMDMs (1 × 10 6 cells per well) were treated with LPS (1 μg/ml) as a 1 st signal for priming, after which cells were replaced with media containing an inflammasome trigger (2 nd signal) with/without heavy metals (HMs) as indicated. Inflammasome activation was assessed by ELISA and/or immunoblotting. ( B ) LPS-primed BMDMs were treated with nigericin (NG) in the presence of media only (Non), cadmium (Cd), mercury (Hg), arsenic (As), or lead (Pb). Release of IL-1β was analyzed by ELISA. ( C ) LPS-primed BMDMs were subjected to activation of NLRP3 inflammasome by NG, monosodium urate crystal (MSU), or aluminum (Alum) treatment in the presence of an increasing dosage of mercury (Hg). The cleaved form of caspase-1 (Casp1, p20) was detected by immunoblotting, and secretion of IL-1β was observed with ELISA. ( D ) LPS-primed BMDMs were treated with NG, MSU, or Alum in the presence of arsenic (As). Release of Casp1 and IL-1β was analyzed with immunoblotting and ELISA. All immunoblot data shown are representative of at least three independent experiments. Bar graph presents the mean ± SD with at least two independent experiments.

    Article Snippet: To activate NLRP3 inflammasome, cells were treated with RPMI 1640 medium (350 μl/well) without FBS or antibiotics in the presence of nigericin (NG, 40 μM; 4312, Tocris Bioscience, Bristol, UK) for 1 h, monosodium urate crystals (MSU, 400 μg/ml; U2875, Sigma–Aldrich Co.) which were prepared in according to a previous study for 3 h, aluminium potassium sulfate (Alum, 200 μg/ml; 039–4404, Daejung Chemicals & Materials Co., Daejeon, Republic of Korea) for 3 h, or rotenone (160 μM; sc-203242, Santa Cruz Biotechnology, Dallas, TX, USA) for 6 h. For non-canonical inflammasome activation, cells were transfected by LPS (15 μg/ml, Sigma-Aldrich Co) with Lipofectamine 2000 (10 μl/ml, Invitrogen) and incubated for 6 h or infected by Escherichia coli (MOI 10, DH5α, Invitrogen, CA, USA) for 3 h. Heavy metals, cadmium acetate dihydreate (CAS No. 5743-04-04, Cat. No. 2616-4140), mercury(II) chloride (CAS No. 7487-94-7, Cat. No. 5542-4425), arsenic(III) oxide (arsenic trioxide, CAS No. 1327-53-3, Cat. No. 5076-1405), lead(II) acetate trihydrate (CAS No. 6080-56-4, Cat. No. 5075-4405), or lead(II) chloride (CAS No. 7758-95-4, Cat. No. 5076-1405), were purchased from Daejung Chemicals & Materials Co. and co-treated with the above inflammasome triggers.

    Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay

    Inhibition of MIF prevents NLRP3 inflammasome activation. ASC-cerulean macrophages were primed with LPS (10 ng ml − 1 ) overnight. The following day cells were treated with COR123625 (50 µM) for 2 h before activation of the inflammasome with a nigericin (10 µM) (1 h) or b silica (150 µg ml − 1 ) (4 h). Confocal images are representative of three independent experiments. c , d Data are presented as the percentage of ASC-speck-positive cells. Data shown are mean ± SEM of three independent experiments. e WT BMDMs were left untreated, treated with LPS alone (100 ng ml − 1 ) for 5 h, treated with COR123625 for 1 h prior to the addition of LPS, primed with LPS before inflammasome activation with nigericin (5 µM) for 1 h, or treated with COR123625 before LPS and nigericin stimulation. Levels of LDH release were quantified using the Promega cytotoxicity assay. f WT or Mif − / − BMDMs were treated with LPS (10 ng ml − 1 ) + nigericin (5 μM) and lysates and supernatants analyzed by Western blot for caspase-1 and IL-1β. Images are representative of > 3 mice. g BMDMs from WT and Mif − / − mice were treated with (10 ng ml − 1 ) + nigericin (5 μM), fixed and stained for ASC, and analyzed by confocal microscopy. Images are z projections of multiple z-stacks. h Quantitation of ASC specks in g , n = 3 mice per group. Data are expressed as percentage increase of mean ± SEM from three mice. * P

    Journal: Nature Communications

    Article Title: Macrophage migration inhibitory factor is required for NLRP3 inflammasome activation

    doi: 10.1038/s41467-018-04581-2

    Figure Lengend Snippet: Inhibition of MIF prevents NLRP3 inflammasome activation. ASC-cerulean macrophages were primed with LPS (10 ng ml − 1 ) overnight. The following day cells were treated with COR123625 (50 µM) for 2 h before activation of the inflammasome with a nigericin (10 µM) (1 h) or b silica (150 µg ml − 1 ) (4 h). Confocal images are representative of three independent experiments. c , d Data are presented as the percentage of ASC-speck-positive cells. Data shown are mean ± SEM of three independent experiments. e WT BMDMs were left untreated, treated with LPS alone (100 ng ml − 1 ) for 5 h, treated with COR123625 for 1 h prior to the addition of LPS, primed with LPS before inflammasome activation with nigericin (5 µM) for 1 h, or treated with COR123625 before LPS and nigericin stimulation. Levels of LDH release were quantified using the Promega cytotoxicity assay. f WT or Mif − / − BMDMs were treated with LPS (10 ng ml − 1 ) + nigericin (5 μM) and lysates and supernatants analyzed by Western blot for caspase-1 and IL-1β. Images are representative of > 3 mice. g BMDMs from WT and Mif − / − mice were treated with (10 ng ml − 1 ) + nigericin (5 μM), fixed and stained for ASC, and analyzed by confocal microscopy. Images are z projections of multiple z-stacks. h Quantitation of ASC specks in g , n = 3 mice per group. Data are expressed as percentage increase of mean ± SEM from three mice. * P

    Article Snippet: Cells were then stimulated with inflammasome activators: nigericin (Cayman Chemical) (5 or 10 µM for 5–60 min), adenosine 5′-triphosphate disodium salt (ATP, Sigma) (5 or 10 mM for 1 h), silica (Invivogen) (150 µg ml− 1 for 1–6 h), MSU (Invivogen) (150 µg ml− 1 for 6 h), PBI-F2 peptide (provided by A. Mansell) (100 μg ml− 1 for 6 h).

    Techniques: Inhibition, Activation Assay, Cytotoxicity Assay, Western Blot, Mouse Assay, Staining, Confocal Microscopy, Quantitation Assay

    MIF interacts with NLRP3. a Immortalized BMDMs were left untreated, primed with LPS alone (100 ng ml − 1 ) for 5 h, or primed with LPS before inflammasome activation with nigericin (5 µM, 30 min). Lysates were immunoprecipitated with anti-MIF or anti-NLRP3 antibody, followed by western blot analysis with antibodies against NLRP3 and MIF. b WT BMDMs were primed with LPS alone (10 ng ml − 1 ) for 5 h, primed with LPS followed by NLRP3 activation with nigericin (5 µM) for 30 min, or primed with LPS followed by COR123625 (50 µM) treatment for 2 h prior to the addition of nigericin (5 µM) for 30 min. Levels of interaction between MIF and NLRP3 were assessed by FLIM-FRET microscopy. FLIM-FRET images presented are representative of three independent experiments. Scale bar = 50 μm. Graphs show changes in the amplitude-weighted average lifetime ( τ Av Amp) of the donor (A488) due to proximity to the acceptor (A568). Combined data from three mice, analyzed by paired t test. * P

    Journal: Nature Communications

    Article Title: Macrophage migration inhibitory factor is required for NLRP3 inflammasome activation

    doi: 10.1038/s41467-018-04581-2

    Figure Lengend Snippet: MIF interacts with NLRP3. a Immortalized BMDMs were left untreated, primed with LPS alone (100 ng ml − 1 ) for 5 h, or primed with LPS before inflammasome activation with nigericin (5 µM, 30 min). Lysates were immunoprecipitated with anti-MIF or anti-NLRP3 antibody, followed by western blot analysis with antibodies against NLRP3 and MIF. b WT BMDMs were primed with LPS alone (10 ng ml − 1 ) for 5 h, primed with LPS followed by NLRP3 activation with nigericin (5 µM) for 30 min, or primed with LPS followed by COR123625 (50 µM) treatment for 2 h prior to the addition of nigericin (5 µM) for 30 min. Levels of interaction between MIF and NLRP3 were assessed by FLIM-FRET microscopy. FLIM-FRET images presented are representative of three independent experiments. Scale bar = 50 μm. Graphs show changes in the amplitude-weighted average lifetime ( τ Av Amp) of the donor (A488) due to proximity to the acceptor (A568). Combined data from three mice, analyzed by paired t test. * P

    Article Snippet: Cells were then stimulated with inflammasome activators: nigericin (Cayman Chemical) (5 or 10 µM for 5–60 min), adenosine 5′-triphosphate disodium salt (ATP, Sigma) (5 or 10 mM for 1 h), silica (Invivogen) (150 µg ml− 1 for 1–6 h), MSU (Invivogen) (150 µg ml− 1 for 6 h), PBI-F2 peptide (provided by A. Mansell) (100 μg ml− 1 for 6 h).

    Techniques: Activation Assay, Immunoprecipitation, Western Blot, Microscopy, Mouse Assay

    MIF is required for interactions between NLRP3 and vimentin. a Primary murine WT and Mif −/− BMDMs were left untreated, primed with LPS alone (100 ng ml − 1 ) for 5 h, or primed with LPS before inflammasome activation with nigericin (5 µM) for indicated times. Lysates were immunoprecipitated with anti-NLRP3 antibody, followed by Western blot analysis with anti-vimentin antibody. b WT BMDMs were primed with LPS alone (100 ng ml − 1 ) for 5 h, primed with LPS followed by nigericin (5 µM) for 1 h, or primed with LPS followed by COR123625 (50 µM) for 2 h prior to nigericin (5 µM) treatment for 30 min. Levels of interaction between NLRP3 and vimentin were assessed by FLIM-FRET. FLIM-FRET images presented are representative of three independent experiments (three mice). Scale bar = 50 μm. c Changes in the amplitude-weighted average lifetime ( τ Av Amp) of the donor (A488) due to proximity to the acceptor (A568). Representative graph from one mouse (nine separate fields per mouse). **** P

    Journal: Nature Communications

    Article Title: Macrophage migration inhibitory factor is required for NLRP3 inflammasome activation

    doi: 10.1038/s41467-018-04581-2

    Figure Lengend Snippet: MIF is required for interactions between NLRP3 and vimentin. a Primary murine WT and Mif −/− BMDMs were left untreated, primed with LPS alone (100 ng ml − 1 ) for 5 h, or primed with LPS before inflammasome activation with nigericin (5 µM) for indicated times. Lysates were immunoprecipitated with anti-NLRP3 antibody, followed by Western blot analysis with anti-vimentin antibody. b WT BMDMs were primed with LPS alone (100 ng ml − 1 ) for 5 h, primed with LPS followed by nigericin (5 µM) for 1 h, or primed with LPS followed by COR123625 (50 µM) for 2 h prior to nigericin (5 µM) treatment for 30 min. Levels of interaction between NLRP3 and vimentin were assessed by FLIM-FRET. FLIM-FRET images presented are representative of three independent experiments (three mice). Scale bar = 50 μm. c Changes in the amplitude-weighted average lifetime ( τ Av Amp) of the donor (A488) due to proximity to the acceptor (A568). Representative graph from one mouse (nine separate fields per mouse). **** P

    Article Snippet: Cells were then stimulated with inflammasome activators: nigericin (Cayman Chemical) (5 or 10 µM for 5–60 min), adenosine 5′-triphosphate disodium salt (ATP, Sigma) (5 or 10 mM for 1 h), silica (Invivogen) (150 µg ml− 1 for 1–6 h), MSU (Invivogen) (150 µg ml− 1 for 6 h), PBI-F2 peptide (provided by A. Mansell) (100 μg ml− 1 for 6 h).

    Techniques: Activation Assay, Immunoprecipitation, Western Blot, Mouse Assay

    MIF co-localizes with the NLRP3 inflammasome. a ASC-cerulean macrophages were primed with LPS (10 ng ml − 1 ) overnight. The following day cells were treated with nigericin (10 µM) for the indicated times. Co-localization of ASC (green) and MIF (red) were visualized using confocal microscopy. Confocal images are representative of at least three independent experiments. b Primary murine BMDMs were treated with LPS (10 ng ml − 1 ) overnight, and then treated with or without COR123625 (50 µM), followed by nigericin (5 µM) for 20 min. Cells were fixed and stained with antibodies against NLRP3 and MIF. Nuclei were stained with DAPI. All scale bars = 10 μm

    Journal: Nature Communications

    Article Title: Macrophage migration inhibitory factor is required for NLRP3 inflammasome activation

    doi: 10.1038/s41467-018-04581-2

    Figure Lengend Snippet: MIF co-localizes with the NLRP3 inflammasome. a ASC-cerulean macrophages were primed with LPS (10 ng ml − 1 ) overnight. The following day cells were treated with nigericin (10 µM) for the indicated times. Co-localization of ASC (green) and MIF (red) were visualized using confocal microscopy. Confocal images are representative of at least three independent experiments. b Primary murine BMDMs were treated with LPS (10 ng ml − 1 ) overnight, and then treated with or without COR123625 (50 µM), followed by nigericin (5 µM) for 20 min. Cells were fixed and stained with antibodies against NLRP3 and MIF. Nuclei were stained with DAPI. All scale bars = 10 μm

    Article Snippet: Cells were then stimulated with inflammasome activators: nigericin (Cayman Chemical) (5 or 10 µM for 5–60 min), adenosine 5′-triphosphate disodium salt (ATP, Sigma) (5 or 10 mM for 1 h), silica (Invivogen) (150 µg ml− 1 for 1–6 h), MSU (Invivogen) (150 µg ml− 1 for 6 h), PBI-F2 peptide (provided by A. Mansell) (100 μg ml− 1 for 6 h).

    Techniques: Confocal Microscopy, Staining

    MIF is required for the release of IL-1 family cytokines. Primary murine WT and Mif −/− BMDMs were left untreated (control), primed with LPS (10 ng ml − 1 ), or primed with LPS followed by nigericin (5 µM) treatment for 1 h. Levels of a IL-1α, b IL-1β, and c IL-18 in cell culture supernatants were assessed by ELISA. Primary WT BMDMs were left untreated, primed with LPS alone (10 ng ml − 1 ), primed with LPS, and then treated with or without COR123625 (50 µM) for 2 h before the addition of nigericin (5 µM) for 1 h. Levels of d IL-1α, e IL-1β, and f IL-18 in cell culture supernatants were assessed by ELISA. C57BL6/J mice were injected intraperitoneally with vehicle control (saline), LPS alone (2 mg kg − 1 ) or COR123625 (20 mg kg − 1 ) in combination with LPS for 2 h. Serum levels of g IL-1β, h IL-18, and i IL-6 were measured by ELISA. Data are expressed as means ± SEM, n = 3–4 mice per group. * P

    Journal: Nature Communications

    Article Title: Macrophage migration inhibitory factor is required for NLRP3 inflammasome activation

    doi: 10.1038/s41467-018-04581-2

    Figure Lengend Snippet: MIF is required for the release of IL-1 family cytokines. Primary murine WT and Mif −/− BMDMs were left untreated (control), primed with LPS (10 ng ml − 1 ), or primed with LPS followed by nigericin (5 µM) treatment for 1 h. Levels of a IL-1α, b IL-1β, and c IL-18 in cell culture supernatants were assessed by ELISA. Primary WT BMDMs were left untreated, primed with LPS alone (10 ng ml − 1 ), primed with LPS, and then treated with or without COR123625 (50 µM) for 2 h before the addition of nigericin (5 µM) for 1 h. Levels of d IL-1α, e IL-1β, and f IL-18 in cell culture supernatants were assessed by ELISA. C57BL6/J mice were injected intraperitoneally with vehicle control (saline), LPS alone (2 mg kg − 1 ) or COR123625 (20 mg kg − 1 ) in combination with LPS for 2 h. Serum levels of g IL-1β, h IL-18, and i IL-6 were measured by ELISA. Data are expressed as means ± SEM, n = 3–4 mice per group. * P

    Article Snippet: Cells were then stimulated with inflammasome activators: nigericin (Cayman Chemical) (5 or 10 µM for 5–60 min), adenosine 5′-triphosphate disodium salt (ATP, Sigma) (5 or 10 mM for 1 h), silica (Invivogen) (150 µg ml− 1 for 1–6 h), MSU (Invivogen) (150 µg ml− 1 for 6 h), PBI-F2 peptide (provided by A. Mansell) (100 μg ml− 1 for 6 h).

    Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Mouse Assay, Injection

    MIF regulates NLRP3-dependent release of IL-1 family cytokines. a Primary murine WT or Mif −/− BMDMs were left untreated, primed with LPS alone (10 ng ml − 1 ), or primed with LPS before transfection of poly dA:dT (1 µg ml − 1 ) for 5 h. WT BMDMs were left untreated, primed with LPS (10 ng ml − 1 ), or primed with LPS before the addition of COR123625 (50 µM) for 2 h before transfection of b poly dA:dT (1 µg ml − 1 ) or c flagellin (250 ng ml − 1 ) for 5 h. Alternatively, following priming of WT BMDMs with LPS, cells were treated with d , g MSU (150 µg ml − 1 ), e , h silica (150 µg ml − 1 ), or ( f , i ) PBI-F2 peptide (100 µg ml − 1 ) for 6 h. Levels of a – f IL-1β and g – i IL-18 in cell culture supernatants were quantified by ELISA. j Primary WT or Mif −/− BMDMs were primed with LPS (10 ng ml − 1 ) in the presence or absence of recombinant murine MIF (rMIF, 10 ng ml − 1 ) before stimulation with nigericin (5 µM) for 1 h. In addition, cells were treated with or without COR123625. Levels of IL-1β in cell culture supernatants were assessed by ELISA. k Primary WT or Mif − / − BMDMs were primed with LPS (10 ng ml − 1 ) in the presence or absence of MIF-containing conditioned media from WT BMDM before stimulation with nigericin (5 µM) for 1 h. Levels of IL-1β in cell culture supernatants were assessed by ELISA. Data are expressed as means ± SEM, n = 3 mice. *P

    Journal: Nature Communications

    Article Title: Macrophage migration inhibitory factor is required for NLRP3 inflammasome activation

    doi: 10.1038/s41467-018-04581-2

    Figure Lengend Snippet: MIF regulates NLRP3-dependent release of IL-1 family cytokines. a Primary murine WT or Mif −/− BMDMs were left untreated, primed with LPS alone (10 ng ml − 1 ), or primed with LPS before transfection of poly dA:dT (1 µg ml − 1 ) for 5 h. WT BMDMs were left untreated, primed with LPS (10 ng ml − 1 ), or primed with LPS before the addition of COR123625 (50 µM) for 2 h before transfection of b poly dA:dT (1 µg ml − 1 ) or c flagellin (250 ng ml − 1 ) for 5 h. Alternatively, following priming of WT BMDMs with LPS, cells were treated with d , g MSU (150 µg ml − 1 ), e , h silica (150 µg ml − 1 ), or ( f , i ) PBI-F2 peptide (100 µg ml − 1 ) for 6 h. Levels of a – f IL-1β and g – i IL-18 in cell culture supernatants were quantified by ELISA. j Primary WT or Mif −/− BMDMs were primed with LPS (10 ng ml − 1 ) in the presence or absence of recombinant murine MIF (rMIF, 10 ng ml − 1 ) before stimulation with nigericin (5 µM) for 1 h. In addition, cells were treated with or without COR123625. Levels of IL-1β in cell culture supernatants were assessed by ELISA. k Primary WT or Mif − / − BMDMs were primed with LPS (10 ng ml − 1 ) in the presence or absence of MIF-containing conditioned media from WT BMDM before stimulation with nigericin (5 µM) for 1 h. Levels of IL-1β in cell culture supernatants were assessed by ELISA. Data are expressed as means ± SEM, n = 3 mice. *P

    Article Snippet: Cells were then stimulated with inflammasome activators: nigericin (Cayman Chemical) (5 or 10 µM for 5–60 min), adenosine 5′-triphosphate disodium salt (ATP, Sigma) (5 or 10 mM for 1 h), silica (Invivogen) (150 µg ml− 1 for 1–6 h), MSU (Invivogen) (150 µg ml− 1 for 6 h), PBI-F2 peptide (provided by A. Mansell) (100 μg ml− 1 for 6 h).

    Techniques: Transfection, Cell Culture, Enzyme-linked Immunosorbent Assay, Recombinant, Mouse Assay

    MARK4 interacts with NLRP3 in a microtubule-dependent manner. ( a ) Upon nigericin stimulation (3 μM for 2 h), microtubule-disrupting drugs colchicine and nocodazole reduced the interaction between Mark4 and Nlrp3, shown by PLA signals in WT BMDM cells. Mark4 KO and Nlrp3 KO BMDM cells were employed as controls. ( b ) MARK4 was associated with NLRP3 in co-immunoprecipitation assay. Whole cell lysates were analysed as indication of transfection. Western blots are representative of 3 independent experiments. ( c ) Upon nigericin stimulation, PLA signal of NlrpP3 and Mark4, or Asc and Mark4 in BMDM cells derived from WT or Nlrp3 KO. Secondary only was employed as control in this PLA assay. ( d ) PLA signal of Nlrp3 and Asc in BMDM cells derived from WT or Nlrp3 KO or Asc KO before or after nigericin stimulation (3 μM for 2 h). Mean±s.e.m. for all the cells taken from 5 to 8 different views at × 40 magnification for each group ( a , b , d ). Comparisons of the two different groups were analysed by unpaired t -test. NS was considered as not statistically significant. * P

    Journal: Nature Communications

    Article Title: MARK4 regulates NLRP3 positioning and inflammasome activation through a microtubule-dependent mechanism

    doi: 10.1038/ncomms15986

    Figure Lengend Snippet: MARK4 interacts with NLRP3 in a microtubule-dependent manner. ( a ) Upon nigericin stimulation (3 μM for 2 h), microtubule-disrupting drugs colchicine and nocodazole reduced the interaction between Mark4 and Nlrp3, shown by PLA signals in WT BMDM cells. Mark4 KO and Nlrp3 KO BMDM cells were employed as controls. ( b ) MARK4 was associated with NLRP3 in co-immunoprecipitation assay. Whole cell lysates were analysed as indication of transfection. Western blots are representative of 3 independent experiments. ( c ) Upon nigericin stimulation, PLA signal of NlrpP3 and Mark4, or Asc and Mark4 in BMDM cells derived from WT or Nlrp3 KO. Secondary only was employed as control in this PLA assay. ( d ) PLA signal of Nlrp3 and Asc in BMDM cells derived from WT or Nlrp3 KO or Asc KO before or after nigericin stimulation (3 μM for 2 h). Mean±s.e.m. for all the cells taken from 5 to 8 different views at × 40 magnification for each group ( a , b , d ). Comparisons of the two different groups were analysed by unpaired t -test. NS was considered as not statistically significant. * P

    Article Snippet: Nigericin, flagellin, poly (dA:dT) and vinblastine were obtained from Enzo. iE-DAP was obtained from Invivogen.

    Techniques: Proximity Ligation Assay, Co-Immunoprecipitation Assay, Transfection, Western Blot, Derivative Assay

    MARK4 deficiency affects MTOC and speck nucleation. ( a ) Differentiated THP-1 cells were stimulated with nigericin (10 μΜ for 1.5 h) or left untreated (control). Endogeneous levels of NLRP3 and MARK4 were co-stained with γ-tubulin. ( b , c ) HEK293T cells were co-overexpressed with GFP-MARK4, Cherry-NLRP3 and ASC-Flag (indicated as MARK4 GFP o.e.); or co-overexpressed with MARK4 shRNA (shown by green GFP), Cherry-NLRP3 and ASC-Flag (indicated as MARK4 shRNA). co-overexpression with MARK4-GFP drove NLRP3 to MTOC, and knock down of MARK4 by shRNA (indicated by GFP) led to a dilated ring structure of NLRP3. Quantification of speck size was shown. Scale bar, 40 μm. Mean±s.e.m. for all the cells taken from five different views at × 20 magnification for each group. Comparisons of the two different groups were analysed by unpaired t -test. **** P

    Journal: Nature Communications

    Article Title: MARK4 regulates NLRP3 positioning and inflammasome activation through a microtubule-dependent mechanism

    doi: 10.1038/ncomms15986

    Figure Lengend Snippet: MARK4 deficiency affects MTOC and speck nucleation. ( a ) Differentiated THP-1 cells were stimulated with nigericin (10 μΜ for 1.5 h) or left untreated (control). Endogeneous levels of NLRP3 and MARK4 were co-stained with γ-tubulin. ( b , c ) HEK293T cells were co-overexpressed with GFP-MARK4, Cherry-NLRP3 and ASC-Flag (indicated as MARK4 GFP o.e.); or co-overexpressed with MARK4 shRNA (shown by green GFP), Cherry-NLRP3 and ASC-Flag (indicated as MARK4 shRNA). co-overexpression with MARK4-GFP drove NLRP3 to MTOC, and knock down of MARK4 by shRNA (indicated by GFP) led to a dilated ring structure of NLRP3. Quantification of speck size was shown. Scale bar, 40 μm. Mean±s.e.m. for all the cells taken from five different views at × 20 magnification for each group. Comparisons of the two different groups were analysed by unpaired t -test. **** P

    Article Snippet: Nigericin, flagellin, poly (dA:dT) and vinblastine were obtained from Enzo. iE-DAP was obtained from Invivogen.

    Techniques: Staining, shRNA, Over Expression

    MARK4 is involved in NLRP3 positioning along microtubules. ( a ) Upon nigericin stimulation (3 μM), NLRP3 (arrow) was moving along microtubules (MT) to meet mitochondria (arrowhead) in THP-1 cells stably expressing NLRP3-cherry. Scale bar, 2 μm. See also Supplementary Movie 1 . ( b ) Orthogonal view of NLRP3 with mitochondria in THP-1 cells stably expressing shRNA of MARK4 or scrambled controls. See also Supplementary Movie 2 . ( c ) Cherry-NLRP3 and GFP-MARK4 were moving together towards MTOC in differentiated THP-1 cells upon nigericin stimulation. Histogram mean of MARK4-GFP was calculated within the indicated square. Arrowheads indicate MTOC. See also Supplementary Movie 4 . ( d ) Orthogonal view of NLRP3 with microtubule in THP-1 cells stably expressing NLRP3-Cherry before or after stimulation by nigericin (10 μΜ for 2 h); orthogonal view was centered around MTOC. See also Supplementary Movie 5 . Experiments were repeated at least three times. Scale bar, 10 μm ( b – d ).

    Journal: Nature Communications

    Article Title: MARK4 regulates NLRP3 positioning and inflammasome activation through a microtubule-dependent mechanism

    doi: 10.1038/ncomms15986

    Figure Lengend Snippet: MARK4 is involved in NLRP3 positioning along microtubules. ( a ) Upon nigericin stimulation (3 μM), NLRP3 (arrow) was moving along microtubules (MT) to meet mitochondria (arrowhead) in THP-1 cells stably expressing NLRP3-cherry. Scale bar, 2 μm. See also Supplementary Movie 1 . ( b ) Orthogonal view of NLRP3 with mitochondria in THP-1 cells stably expressing shRNA of MARK4 or scrambled controls. See also Supplementary Movie 2 . ( c ) Cherry-NLRP3 and GFP-MARK4 were moving together towards MTOC in differentiated THP-1 cells upon nigericin stimulation. Histogram mean of MARK4-GFP was calculated within the indicated square. Arrowheads indicate MTOC. See also Supplementary Movie 4 . ( d ) Orthogonal view of NLRP3 with microtubule in THP-1 cells stably expressing NLRP3-Cherry before or after stimulation by nigericin (10 μΜ for 2 h); orthogonal view was centered around MTOC. See also Supplementary Movie 5 . Experiments were repeated at least three times. Scale bar, 10 μm ( b – d ).

    Article Snippet: Nigericin, flagellin, poly (dA:dT) and vinblastine were obtained from Enzo. iE-DAP was obtained from Invivogen.

    Techniques: Stable Transfection, Expressing, shRNA

    CagA impairs inflammasome activation by disrupting NLRP3-MARK4 interaction. ( a ) CagA peptide impaired the interaction between NLRP3 and MARK4 upon NLRP3 inflammasome activation by nigericin (3 μM for 2 h) in the differentiated THP-1 cells. A control peptide was employed. Scale bar, 10 μm. ( b ) ELISA of IL-1β level in the supernatants of WT BMDM cells following pretreatment of CagA peptide (with indicated concentrations) and nigericin stimulation (10 μM for 1 h). Mark4 KO cells were used as controls. ( c ) CagA peptide prevented formation of Nlrp3 and Asc complex under nigericin stimulation (3 μM for 2 h) in WT BMDMs. Mark4 KO cells were used as controls. Scale bar, 10 μm. Mean±s.e.m. for all the cells taken from five different views at × 40 magnification for each group ( a , c ). ( d ) CagA peptide impaired the formation of NLRP3 speck on MTOC upon NLRP3 inflammasome activation (MSU 250 μg ml −1 for 6 h) in the differentiated THP-1 cells. Inset was used to display magnification of the boxed region as examples of MARK4, NLRP3, and γ-tubulin localization. Scale bar, 20 μm. Mean±s.e.m., at least three experiments ( a – d ). Comparisons of the two different groups were analysed by unpaired t test. NS was considered as not statistically significant. * P

    Journal: Nature Communications

    Article Title: MARK4 regulates NLRP3 positioning and inflammasome activation through a microtubule-dependent mechanism

    doi: 10.1038/ncomms15986

    Figure Lengend Snippet: CagA impairs inflammasome activation by disrupting NLRP3-MARK4 interaction. ( a ) CagA peptide impaired the interaction between NLRP3 and MARK4 upon NLRP3 inflammasome activation by nigericin (3 μM for 2 h) in the differentiated THP-1 cells. A control peptide was employed. Scale bar, 10 μm. ( b ) ELISA of IL-1β level in the supernatants of WT BMDM cells following pretreatment of CagA peptide (with indicated concentrations) and nigericin stimulation (10 μM for 1 h). Mark4 KO cells were used as controls. ( c ) CagA peptide prevented formation of Nlrp3 and Asc complex under nigericin stimulation (3 μM for 2 h) in WT BMDMs. Mark4 KO cells were used as controls. Scale bar, 10 μm. Mean±s.e.m. for all the cells taken from five different views at × 40 magnification for each group ( a , c ). ( d ) CagA peptide impaired the formation of NLRP3 speck on MTOC upon NLRP3 inflammasome activation (MSU 250 μg ml −1 for 6 h) in the differentiated THP-1 cells. Inset was used to display magnification of the boxed region as examples of MARK4, NLRP3, and γ-tubulin localization. Scale bar, 20 μm. Mean±s.e.m., at least three experiments ( a – d ). Comparisons of the two different groups were analysed by unpaired t test. NS was considered as not statistically significant. * P

    Article Snippet: Nigericin, flagellin, poly (dA:dT) and vinblastine were obtained from Enzo. iE-DAP was obtained from Invivogen.

    Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay

    ROS is involved in inflammasome activation induced by ER stress. a ROS production detected by flow cytometry in LPS, TG, or LPS plus TG-treated BMDMs (UT unstimulated). b ROS production in LPS plus TG-treated BMDMs with or without Nec-1 pretreatment. c ROS production in LPS plus TG-treated BMDMs transfected with control siRNA (Scr) or RIP1-specific siRNAs. d ROS production in LPS plus TG-treated BMDMs with or without Midivi-1 or BHA pretreatment. e , f Secretion of IL-1β and TNF-α by BMDMs stimulated by LPS plus TG or LPS plus Nigericin with or without antioxidant BHA pretreatment. g Immunoblot of cleaved caspase-1 (p20) in cell supernatants (SN) and caspase-1 (pro caspase-1) and β-actin in cell lysates (Input) of BMDMs stimulated with LPS plus TG or LPS plus Nigericin with or without BHA pretreatment. Figures are representative of at least three independent experiments. Bars indicate means plus SD. ns not significant, ** P

    Journal: Cell Death & Disease

    Article Title: The kinase receptor-interacting protein 1 is required for inflammasome activation induced by endoplasmic reticulum stress

    doi: 10.1038/s41419-018-0694-7

    Figure Lengend Snippet: ROS is involved in inflammasome activation induced by ER stress. a ROS production detected by flow cytometry in LPS, TG, or LPS plus TG-treated BMDMs (UT unstimulated). b ROS production in LPS plus TG-treated BMDMs with or without Nec-1 pretreatment. c ROS production in LPS plus TG-treated BMDMs transfected with control siRNA (Scr) or RIP1-specific siRNAs. d ROS production in LPS plus TG-treated BMDMs with or without Midivi-1 or BHA pretreatment. e , f Secretion of IL-1β and TNF-α by BMDMs stimulated by LPS plus TG or LPS plus Nigericin with or without antioxidant BHA pretreatment. g Immunoblot of cleaved caspase-1 (p20) in cell supernatants (SN) and caspase-1 (pro caspase-1) and β-actin in cell lysates (Input) of BMDMs stimulated with LPS plus TG or LPS plus Nigericin with or without BHA pretreatment. Figures are representative of at least three independent experiments. Bars indicate means plus SD. ns not significant, ** P

    Article Snippet: Nigericin was from Merck-Millipore and Mdivi-1 was from Selleckchem.

    Techniques: Activation Assay, Flow Cytometry, Cytometry, Transfection

    Cell death is not involved in ER stress-induced inflammasome activation. a , c Cell death induced by ER stress inducer TG in BMDMs transfected with control siRNA (Scr) or RIP1-specific siRNAs (siRip1) or DRP1-specific siRNAs (siDRP1). The cell death rate was measured by LDH release assay. b Cell death induced by LPS plus TG treatment or LPS plus Nigericin treatment in WT, RIP3 +/ − , and RIP3 −/− BMDMs. d Cell death induced by LPS plus TG with or without pretreatment of various inhibitors as indicated. Results are from three independent experiments with biological duplicates in each. Bars indicate means plus SD. ns not significant (unpaired Student’s t test)

    Journal: Cell Death & Disease

    Article Title: The kinase receptor-interacting protein 1 is required for inflammasome activation induced by endoplasmic reticulum stress

    doi: 10.1038/s41419-018-0694-7

    Figure Lengend Snippet: Cell death is not involved in ER stress-induced inflammasome activation. a , c Cell death induced by ER stress inducer TG in BMDMs transfected with control siRNA (Scr) or RIP1-specific siRNAs (siRip1) or DRP1-specific siRNAs (siDRP1). The cell death rate was measured by LDH release assay. b Cell death induced by LPS plus TG treatment or LPS plus Nigericin treatment in WT, RIP3 +/ − , and RIP3 −/− BMDMs. d Cell death induced by LPS plus TG with or without pretreatment of various inhibitors as indicated. Results are from three independent experiments with biological duplicates in each. Bars indicate means plus SD. ns not significant (unpaired Student’s t test)

    Article Snippet: Nigericin was from Merck-Millipore and Mdivi-1 was from Selleckchem.

    Techniques: Activation Assay, Transfection, Lactate Dehydrogenase Assay

    ER stress-induced inflammasome activation is severely reduced by RIP1 siRNA silencing. a The expression level of RIP1 in BMDMs transfected with control siRNA with a scrambled sequence (Scr) or RIP1-specific siRNA (two constructs, siRip1(1) or siRip1(2)). b , c Release of IL-1β and TNF-α by BMDMs which were first transfected with control siRNA (Scr) or RIP1-specific siRNAs and then treated with LPS plus TG or LPS plus Nigericin. UT unstimulated. d Immunoblot analysis of cleaved caspase-1 (p20) in cell supernatants (SN) and caspase-1 (pro-casp1) and β-actin in cell lysates (Input) of BMDMs which were first transfected with control siRNA (Scr) or RIP1-specific siRNAs and then treated with LPS plus TG or LPS plus Nigericin. e WT BMDMs were primed with LPS first and then treated with TG for the indicated time. The phosphorylation of RIP1 was determined by western blot with anti-phospho-RIP1 (Ser166) antibody. Figures are representative of at least three independent experiments. Bars indicate means plus SD. ns not significant, ** P

    Journal: Cell Death & Disease

    Article Title: The kinase receptor-interacting protein 1 is required for inflammasome activation induced by endoplasmic reticulum stress

    doi: 10.1038/s41419-018-0694-7

    Figure Lengend Snippet: ER stress-induced inflammasome activation is severely reduced by RIP1 siRNA silencing. a The expression level of RIP1 in BMDMs transfected with control siRNA with a scrambled sequence (Scr) or RIP1-specific siRNA (two constructs, siRip1(1) or siRip1(2)). b , c Release of IL-1β and TNF-α by BMDMs which were first transfected with control siRNA (Scr) or RIP1-specific siRNAs and then treated with LPS plus TG or LPS plus Nigericin. UT unstimulated. d Immunoblot analysis of cleaved caspase-1 (p20) in cell supernatants (SN) and caspase-1 (pro-casp1) and β-actin in cell lysates (Input) of BMDMs which were first transfected with control siRNA (Scr) or RIP1-specific siRNAs and then treated with LPS plus TG or LPS plus Nigericin. e WT BMDMs were primed with LPS first and then treated with TG for the indicated time. The phosphorylation of RIP1 was determined by western blot with anti-phospho-RIP1 (Ser166) antibody. Figures are representative of at least three independent experiments. Bars indicate means plus SD. ns not significant, ** P

    Article Snippet: Nigericin was from Merck-Millipore and Mdivi-1 was from Selleckchem.

    Techniques: Activation Assay, Expressing, Transfection, Sequencing, Construct, Western Blot

    DRP1 contributes to ER stress-induced inflammasome activation. a The expression level of DRP1 in J774A.1 macrophages transfected with control siRNA with a scrambled sequence (Scr) or DRP1-specific siRNA. b , c Release of IL-1β and TNF-α by J774A.1 macrophages which were first transfected with control siRNA (Scr) or DRP1-specific siRNA and then treated with LPS plus TG or LPS plus Nigericin. d Immunoblot analysis of cleaved caspase-1 (p20) in cell supernatants (SN) and caspase-1 (pro-casp1) and β-actin in cell lysates (Input) of J774A.1 macrophages which were first transfected with control siRNA (Scr) or DRP1-specific siRNA and then treated with LPS plus TG or LPS plus Nigericin. e , f Secretion of IL-1β and TNF-α by BMDMs stimulated by LPS plus TG or LPS plus Nigericin with or without DRP1 inhibitor Mdivi-1 pretreatment. g Immunoblot of cleaved caspase-1 (p20) in cell supernatants (SN) and caspase-1 (pro-casp1) and β-actin in cell lysates (Input) of BMDMs stimulated with LPS plus TG or LPS plus Nigericin with or without Mdivi-1 pretreatment. UT, unstimulated. h Confocal microscopy analysis of BMDMs with or without Nec-1 pretreatment and then stimulated with LPS plus TG. Cells were stained with MitoTracker. Scale bar, 25 μm. Figures are representative of at least three independent experiments. Bars indicate means plus SD. ns not significant, ** P

    Journal: Cell Death & Disease

    Article Title: The kinase receptor-interacting protein 1 is required for inflammasome activation induced by endoplasmic reticulum stress

    doi: 10.1038/s41419-018-0694-7

    Figure Lengend Snippet: DRP1 contributes to ER stress-induced inflammasome activation. a The expression level of DRP1 in J774A.1 macrophages transfected with control siRNA with a scrambled sequence (Scr) or DRP1-specific siRNA. b , c Release of IL-1β and TNF-α by J774A.1 macrophages which were first transfected with control siRNA (Scr) or DRP1-specific siRNA and then treated with LPS plus TG or LPS plus Nigericin. d Immunoblot analysis of cleaved caspase-1 (p20) in cell supernatants (SN) and caspase-1 (pro-casp1) and β-actin in cell lysates (Input) of J774A.1 macrophages which were first transfected with control siRNA (Scr) or DRP1-specific siRNA and then treated with LPS plus TG or LPS plus Nigericin. e , f Secretion of IL-1β and TNF-α by BMDMs stimulated by LPS plus TG or LPS plus Nigericin with or without DRP1 inhibitor Mdivi-1 pretreatment. g Immunoblot of cleaved caspase-1 (p20) in cell supernatants (SN) and caspase-1 (pro-casp1) and β-actin in cell lysates (Input) of BMDMs stimulated with LPS plus TG or LPS plus Nigericin with or without Mdivi-1 pretreatment. UT, unstimulated. h Confocal microscopy analysis of BMDMs with or without Nec-1 pretreatment and then stimulated with LPS plus TG. Cells were stained with MitoTracker. Scale bar, 25 μm. Figures are representative of at least three independent experiments. Bars indicate means plus SD. ns not significant, ** P

    Article Snippet: Nigericin was from Merck-Millipore and Mdivi-1 was from Selleckchem.

    Techniques: Activation Assay, Expressing, Transfection, Sequencing, Confocal Microscopy, Staining

    ER stress-induced inflammasome activation does not depend on RIP3. a , b Secretion of IL-1β and TNF-α by WT, RIP3 +/− , and RIP3 −/− BMDMs stimulated with LPS plus TG or LPS plus Nigericin. UT unstimulated. c Immunoblot analysis of cleaved caspase-1 (p20) in cell supernatants (SN) and caspase-1 (pro-casp1) and β-actin in cell lysates (Input) of WT, RIP3 +/− , and RIP3 −/− BMDMs stimulated with LPS plus TG or LPS plus Nigericin. Figures are representative of at least three independent experiments. Bars indicate means plus SD. ns not significant (unpaired Student’s t test)

    Journal: Cell Death & Disease

    Article Title: The kinase receptor-interacting protein 1 is required for inflammasome activation induced by endoplasmic reticulum stress

    doi: 10.1038/s41419-018-0694-7

    Figure Lengend Snippet: ER stress-induced inflammasome activation does not depend on RIP3. a , b Secretion of IL-1β and TNF-α by WT, RIP3 +/− , and RIP3 −/− BMDMs stimulated with LPS plus TG or LPS plus Nigericin. UT unstimulated. c Immunoblot analysis of cleaved caspase-1 (p20) in cell supernatants (SN) and caspase-1 (pro-casp1) and β-actin in cell lysates (Input) of WT, RIP3 +/− , and RIP3 −/− BMDMs stimulated with LPS plus TG or LPS plus Nigericin. Figures are representative of at least three independent experiments. Bars indicate means plus SD. ns not significant (unpaired Student’s t test)

    Article Snippet: Nigericin was from Merck-Millipore and Mdivi-1 was from Selleckchem.

    Techniques: Activation Assay

    Extracellular specks represent a cell-derived danger signal. ( a ) Confocal imaging of iMøs incubated with in vitro -assembled ASC-mCerulean specks for 2 hours. Scale bar 9 μm. ( b ) Immunoblot of ASC-mCerulean (anti-GFP) in lysates of BMDMs incubated with ASC-mCerulean specks. Non-phagocyted specks were washed away. ( c ) Confocal imaging of iMøs incubated with Dextran-A488 (20 μg/ml) alone or together with the lysosomal damaging LeuLeu-O-Me (0.5 μM), or ASC-mCerulean specks (100 μg/ml) for 4 hours. Scale bars: 7.0 μm top left, 4.9 μm top right, 6.0 μm bottom left, and 2.9 μm bottom right. ( d ) Average size of lysosomes in cells treated as indicated. ( e ) Percentage of recipient ASC-mCherry cells containing ASC-mCherry specks after incubation with silica crystals (100 μg/ml), nigericin (10 μM) or ASC-mCerulean specks (100 μg/ml). ( f-g ) ELISA of IL-1β in supernatants of LPS-primed (200 ng/ml, 2 h) BMDMs that were either left untreated or stimulated with cholesterol crystals (250 μg/ml), ASC specks, or a mock preparation from Pycard −/− iMøs (200 μg/ml). Data are representative from three independent experiments ( a - e ); d , mean + SD of combined data from two independent experiments, in which at least 5 fields/condition were analyzed (Mann-Whitney test, **P

    Journal: Nature immunology

    Article Title: ASC has extracellular and prionoid activities that propagate inflammation

    doi: 10.1038/ni.2913

    Figure Lengend Snippet: Extracellular specks represent a cell-derived danger signal. ( a ) Confocal imaging of iMøs incubated with in vitro -assembled ASC-mCerulean specks for 2 hours. Scale bar 9 μm. ( b ) Immunoblot of ASC-mCerulean (anti-GFP) in lysates of BMDMs incubated with ASC-mCerulean specks. Non-phagocyted specks were washed away. ( c ) Confocal imaging of iMøs incubated with Dextran-A488 (20 μg/ml) alone or together with the lysosomal damaging LeuLeu-O-Me (0.5 μM), or ASC-mCerulean specks (100 μg/ml) for 4 hours. Scale bars: 7.0 μm top left, 4.9 μm top right, 6.0 μm bottom left, and 2.9 μm bottom right. ( d ) Average size of lysosomes in cells treated as indicated. ( e ) Percentage of recipient ASC-mCherry cells containing ASC-mCherry specks after incubation with silica crystals (100 μg/ml), nigericin (10 μM) or ASC-mCerulean specks (100 μg/ml). ( f-g ) ELISA of IL-1β in supernatants of LPS-primed (200 ng/ml, 2 h) BMDMs that were either left untreated or stimulated with cholesterol crystals (250 μg/ml), ASC specks, or a mock preparation from Pycard −/− iMøs (200 μg/ml). Data are representative from three independent experiments ( a - e ); d , mean + SD of combined data from two independent experiments, in which at least 5 fields/condition were analyzed (Mann-Whitney test, **P

    Article Snippet: For the activity assay of ASC specks in supernatants of activated WT BMDMs, cell-free supernatants (40 μl) of LPS-primed (500 ng/ml, 3 h) and ATP (2.5 mM, 40 min), or nigericin (5 μM, 40 min) activated BMDMs were incubated with 10 μl of serum free DMEM, or 10 μl of in vitro -assembled fluorescent ASC specks (1 mg/ml generated from ASC-mCerulean expressing WT or Casp1 −/− iMøs) for 1 h. The reaction mixtures were then fractionated by SDS-PAGE and analyzed by immunoblotting with anti-caspase-1 (Adipogen) or anti-IL-1β (R & D Systems) Abs.

    Techniques: Derivative Assay, Imaging, Incubation, In Vitro, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

    ASC has prionoid features. Confocal imaging of ASC-mCherry expressing iMøs incubated overnight ( a ), or 36 hours ( b ) with in vitro -assembled ASC-mCerulean specks. Open triangles indicate ASC-mCerulean specks that recruited ASC-mCherry and closed triangle indicate ASC-mCerulan specks that have not recruited ASC-mCherry. Scale bars: 2.6 μm. ( c ) Confocal imaging of untreated or LPS-primed, poly dAdT-activated ASC-mCerulean expressing iMøs. Scale bar: 4.9 μm. ( d ) Stimulated emission depletion (STED) microscopy of ASC specks purified from ASC-mCerulean expessing iMøs stimulated with ATP and stained with anti-GFP and Atto 647N-conjugated secondary Abs. Scale bar: 0.5 μm. ( e ) Electron microscopy (EM) of ASC specks prepared from WT or mock preparations from Pycard −/− iMøs. Scale bars: 100 nm. ( f ) EM of in vitro- assembled ASC-mCerulean specks. Scale bars: 0.5 μm. ( g ) EM of ASC-mCerulean specks isolated from LPS-primed, nigericin activated iMøs stained with anti-GFP Abs directly conjugated to 10 nm gold nanoparticles. Data ( a-g ) are representative of at least two independent experiments.

    Journal: Nature immunology

    Article Title: ASC has extracellular and prionoid activities that propagate inflammation

    doi: 10.1038/ni.2913

    Figure Lengend Snippet: ASC has prionoid features. Confocal imaging of ASC-mCherry expressing iMøs incubated overnight ( a ), or 36 hours ( b ) with in vitro -assembled ASC-mCerulean specks. Open triangles indicate ASC-mCerulean specks that recruited ASC-mCherry and closed triangle indicate ASC-mCerulan specks that have not recruited ASC-mCherry. Scale bars: 2.6 μm. ( c ) Confocal imaging of untreated or LPS-primed, poly dAdT-activated ASC-mCerulean expressing iMøs. Scale bar: 4.9 μm. ( d ) Stimulated emission depletion (STED) microscopy of ASC specks purified from ASC-mCerulean expessing iMøs stimulated with ATP and stained with anti-GFP and Atto 647N-conjugated secondary Abs. Scale bar: 0.5 μm. ( e ) Electron microscopy (EM) of ASC specks prepared from WT or mock preparations from Pycard −/− iMøs. Scale bars: 100 nm. ( f ) EM of in vitro- assembled ASC-mCerulean specks. Scale bars: 0.5 μm. ( g ) EM of ASC-mCerulean specks isolated from LPS-primed, nigericin activated iMøs stained with anti-GFP Abs directly conjugated to 10 nm gold nanoparticles. Data ( a-g ) are representative of at least two independent experiments.

    Article Snippet: For the activity assay of ASC specks in supernatants of activated WT BMDMs, cell-free supernatants (40 μl) of LPS-primed (500 ng/ml, 3 h) and ATP (2.5 mM, 40 min), or nigericin (5 μM, 40 min) activated BMDMs were incubated with 10 μl of serum free DMEM, or 10 μl of in vitro -assembled fluorescent ASC specks (1 mg/ml generated from ASC-mCerulean expressing WT or Casp1 −/− iMøs) for 1 h. The reaction mixtures were then fractionated by SDS-PAGE and analyzed by immunoblotting with anti-caspase-1 (Adipogen) or anti-IL-1β (R & D Systems) Abs.

    Techniques: Imaging, Expressing, Incubation, In Vitro, Microscopy, Purification, Staining, Electron Microscopy, Isolation

    Extracellular ASC specks are formed in vivo and accumulate during human chronic inflammatory disease. ( a ) Confocal imaging of subcapsular sinus macrophages of popliteal draining LNs from P. aeruginosa infected mice injected 4 h later via the same route with 4 μg of PE-labeled anti-ASC Abs. Scale bars: 53 μm left panels 37 μm right panels, 9 μm inserts. ( b ) Flow cytometry of ASC-mCerulean specks pre-incubated with two monoclonal anti-ASC Abs (top histograms) or IgG1 isotype controls (bottom histograms) directly labeled with A488 and A647 fluorophores. Amount of specks per μl of sample was determined by subtracting A488 + A647 + events in anti-ASC stained sample from those in IgG1 control stained samples. ( c ) Adjusted quantification of A488 + A647 + ASC specks in cell-free BALF from WT BALB/c mice exposed to cigarette smoke or normal air for 8 weeks. ( d ) Flow cytometry of BALF samples from patients with COPD (5 ml/patient) before and after removal of dead cells by centrifugation (400 x g, 5 min) and size filtration (5 μm). ( e ) Immunoblotting for ASC (right) of cell-free filtered BALF samples from patients with COPD (n = 4), Pneumonia (n = 4), Pulmunary hypertension (n =2) or healthy donors (n =2) after chemical crosslinking with 1 mM of DSS. Cell-free sups from untreated (–) or LPS-primed, nigericin-activated THP-1s were used as controls. Data show representative images ( a ) flow cytometry ( b, d ), and immunoblotting ( e ) analysis from one out of two independent experiments; ( c ) cumulative from one experiment; each symbol represents an individual mouse; horizontal and vertical lines indicate mean + SD, *P = 0.0317 , Mann-Whitney test.

    Journal: Nature immunology

    Article Title: ASC has extracellular and prionoid activities that propagate inflammation

    doi: 10.1038/ni.2913

    Figure Lengend Snippet: Extracellular ASC specks are formed in vivo and accumulate during human chronic inflammatory disease. ( a ) Confocal imaging of subcapsular sinus macrophages of popliteal draining LNs from P. aeruginosa infected mice injected 4 h later via the same route with 4 μg of PE-labeled anti-ASC Abs. Scale bars: 53 μm left panels 37 μm right panels, 9 μm inserts. ( b ) Flow cytometry of ASC-mCerulean specks pre-incubated with two monoclonal anti-ASC Abs (top histograms) or IgG1 isotype controls (bottom histograms) directly labeled with A488 and A647 fluorophores. Amount of specks per μl of sample was determined by subtracting A488 + A647 + events in anti-ASC stained sample from those in IgG1 control stained samples. ( c ) Adjusted quantification of A488 + A647 + ASC specks in cell-free BALF from WT BALB/c mice exposed to cigarette smoke or normal air for 8 weeks. ( d ) Flow cytometry of BALF samples from patients with COPD (5 ml/patient) before and after removal of dead cells by centrifugation (400 x g, 5 min) and size filtration (5 μm). ( e ) Immunoblotting for ASC (right) of cell-free filtered BALF samples from patients with COPD (n = 4), Pneumonia (n = 4), Pulmunary hypertension (n =2) or healthy donors (n =2) after chemical crosslinking with 1 mM of DSS. Cell-free sups from untreated (–) or LPS-primed, nigericin-activated THP-1s were used as controls. Data show representative images ( a ) flow cytometry ( b, d ), and immunoblotting ( e ) analysis from one out of two independent experiments; ( c ) cumulative from one experiment; each symbol represents an individual mouse; horizontal and vertical lines indicate mean + SD, *P = 0.0317 , Mann-Whitney test.

    Article Snippet: For the activity assay of ASC specks in supernatants of activated WT BMDMs, cell-free supernatants (40 μl) of LPS-primed (500 ng/ml, 3 h) and ATP (2.5 mM, 40 min), or nigericin (5 μM, 40 min) activated BMDMs were incubated with 10 μl of serum free DMEM, or 10 μl of in vitro -assembled fluorescent ASC specks (1 mg/ml generated from ASC-mCerulean expressing WT or Casp1 −/− iMøs) for 1 h. The reaction mixtures were then fractionated by SDS-PAGE and analyzed by immunoblotting with anti-caspase-1 (Adipogen) or anti-IL-1β (R & D Systems) Abs.

    Techniques: In Vivo, Imaging, Infection, Mouse Assay, Injection, Labeling, Flow Cytometry, Cytometry, Incubation, Staining, Centrifugation, Filtration, MANN-WHITNEY

    ASC specks accumulate in the extracellular space after inflammasome activation. ( a ) Confocal imaging of untreated or ATP-activated (5 mM, 40 min) ASC-mCerulean expressing iMøs, Scale bar: 22 μm. ( b ) Ex-vivo immunostaining of the subcapsular sinus from permeabilized sections of popliteal LNs from mice 4 h after injection with PBS, or P. aeruginosa. Scale bar: 15 μm and 19 μm. ( c ) Confocal imaging of ASC-mCerulean iMøs treated as in ( a ). Arrows indicate extracellular ASC specks. Scale bars: 4.4 μm and 3 μm. ( d ) Fluorescence imaging of LPS-primed (1 μg/ml) and nigericin-activated (10 μM) ASC-mCerulean expressing THP-1 monocytes. Arrows indicate extracellular ASC specks. Scale bar: 10 μm. ( e ) Flow cytometry and ( i ) quantification of extracellular specks in cell-free supernatants of ASC-mCerulean THP-1s treated as in d . ( f ) Immunoblot of DSS cross-linked endogenous ASC in cell pellets, or 5 μm filtrates of cell-free supernatants from WT THP-1 monocytes treated as in d . ( g ) Quantification of extracellular specks and ( h ) immunoblot of ASC in LPS-primed (250 ng/ml, 3h) iMøs treated as indicated. ( i ) Assessment of LDH release in cell-free supernatants of ASC-mCerulean THP-1s treated as in d , calculated from cells treated with 1% Triton X100. ( j ) Confocal time lapse of ASC-mCerulean THP-1 monocytes treated as in d in the presence of propidium iodide (1 μg/ml). Scale bars 8 μm, time (min). ( k ) Confocal time lapse of ATP-activated ASC-mCerulean iMøs in presence of directly conjugated anti-GFP Alexa 555 mAb (1 μg). Scale bars 33.0 μm, time (min). Data are representative of three ( a , c-e,i ) or two independent experiments ( b , f - h , j , k ); ( e )Technical triplicates (colored lines) from one representative of three independent experiments; (i) combine data from two independent experiments (mean + SD of the number of specks per μl of acquired sample); g , mean + SD of triplicates from a representative of two independent experiments.

    Journal: Nature immunology

    Article Title: ASC has extracellular and prionoid activities that propagate inflammation

    doi: 10.1038/ni.2913

    Figure Lengend Snippet: ASC specks accumulate in the extracellular space after inflammasome activation. ( a ) Confocal imaging of untreated or ATP-activated (5 mM, 40 min) ASC-mCerulean expressing iMøs, Scale bar: 22 μm. ( b ) Ex-vivo immunostaining of the subcapsular sinus from permeabilized sections of popliteal LNs from mice 4 h after injection with PBS, or P. aeruginosa. Scale bar: 15 μm and 19 μm. ( c ) Confocal imaging of ASC-mCerulean iMøs treated as in ( a ). Arrows indicate extracellular ASC specks. Scale bars: 4.4 μm and 3 μm. ( d ) Fluorescence imaging of LPS-primed (1 μg/ml) and nigericin-activated (10 μM) ASC-mCerulean expressing THP-1 monocytes. Arrows indicate extracellular ASC specks. Scale bar: 10 μm. ( e ) Flow cytometry and ( i ) quantification of extracellular specks in cell-free supernatants of ASC-mCerulean THP-1s treated as in d . ( f ) Immunoblot of DSS cross-linked endogenous ASC in cell pellets, or 5 μm filtrates of cell-free supernatants from WT THP-1 monocytes treated as in d . ( g ) Quantification of extracellular specks and ( h ) immunoblot of ASC in LPS-primed (250 ng/ml, 3h) iMøs treated as indicated. ( i ) Assessment of LDH release in cell-free supernatants of ASC-mCerulean THP-1s treated as in d , calculated from cells treated with 1% Triton X100. ( j ) Confocal time lapse of ASC-mCerulean THP-1 monocytes treated as in d in the presence of propidium iodide (1 μg/ml). Scale bars 8 μm, time (min). ( k ) Confocal time lapse of ATP-activated ASC-mCerulean iMøs in presence of directly conjugated anti-GFP Alexa 555 mAb (1 μg). Scale bars 33.0 μm, time (min). Data are representative of three ( a , c-e,i ) or two independent experiments ( b , f - h , j , k ); ( e )Technical triplicates (colored lines) from one representative of three independent experiments; (i) combine data from two independent experiments (mean + SD of the number of specks per μl of acquired sample); g , mean + SD of triplicates from a representative of two independent experiments.

    Article Snippet: For the activity assay of ASC specks in supernatants of activated WT BMDMs, cell-free supernatants (40 μl) of LPS-primed (500 ng/ml, 3 h) and ATP (2.5 mM, 40 min), or nigericin (5 μM, 40 min) activated BMDMs were incubated with 10 μl of serum free DMEM, or 10 μl of in vitro -assembled fluorescent ASC specks (1 mg/ml generated from ASC-mCerulean expressing WT or Casp1 −/− iMøs) for 1 h. The reaction mixtures were then fractionated by SDS-PAGE and analyzed by immunoblotting with anti-caspase-1 (Adipogen) or anti-IL-1β (R & D Systems) Abs.

    Techniques: Activation Assay, Imaging, Expressing, Ex Vivo, Immunostaining, Mouse Assay, Injection, Fluorescence, Flow Cytometry, Cytometry

    Trim21 exposes AdV genomes to cGAS and STING to trigger NLRP3‐dependent inflammasome activation AIM2 mRNA levels from PBMCs, CD19 +ve B cells, CD14 +ve monocytes or HMDM derived from CD14 +ve monocytes were assessed by qPCR, and copy number was determined relative to actin copy number ( n = 5 mean ± s.e.m). HMDM were stimulated with AdV (50,000 pp/cell) and 20 μg/ml h9C12 for 1 h, washed 2× with SFM and then whole cell lysates harvested after a further 5 h. Viral hexon and transgene (GFP) expression in the cytosol was assessed by Western blot. Data are representative of two independent experiments. THP‐1s expressing either a control guide RNA or targeting cGAS and STING were generated and stimulated with 1,000 U/ml IFN‐α for 4 h and protein levels assessed by Western blot. THP‐1s deficient in cGAS and STING were stimulated with AdV (50,000 pp/cell) and 20 μg/ml h9C12, 200 ng/well HT‐DNA, 10 μM Nigericin or 10 ng/ml LPS for 16 h. Data show combined data (mean ± s.e.m) of three experiments with absolute protein values (upper panel) or as % cytokine output of KO cells relative to Ctrl‐treated cells (lower panel), * P ≤ 0.05, *** P ≤ 0.001 unpaired, two‐tailed t ‐test). WT THP‐1s were treated with 5 μM H151 for 30 min before stimulation as in (D). Data in upper panel are representative of three independent experiments. Data in lower panel show combined data of these three experiments (mean ± s.e.m) showing H151‐treated cells relative to media treated cells (* P ≤ 0.05, *** P ≤ 0.001 unpaired, two‐tailed t ‐test). ASC‐GFP THP‐1s were treated with 5 μM H151 for 30 min before stimulation with AdV‐mCherry (50,000 pp/cell) + 20 μg/ml h9C12 or 20 mg/ml IVIg or 200 ng/ well HT‐DNA for 8 h. A representative image (scale bar 50 μm) and quantification of number of cells with ASC specks from one representative experiment of three are shown. Source data are available online for this figure.

    Journal: The EMBO Journal

    Article Title: Antibody and DNA sensing pathways converge to activate the inflammasome during primary human macrophage infection

    doi: 10.15252/embj.2018101365

    Figure Lengend Snippet: Trim21 exposes AdV genomes to cGAS and STING to trigger NLRP3‐dependent inflammasome activation AIM2 mRNA levels from PBMCs, CD19 +ve B cells, CD14 +ve monocytes or HMDM derived from CD14 +ve monocytes were assessed by qPCR, and copy number was determined relative to actin copy number ( n = 5 mean ± s.e.m). HMDM were stimulated with AdV (50,000 pp/cell) and 20 μg/ml h9C12 for 1 h, washed 2× with SFM and then whole cell lysates harvested after a further 5 h. Viral hexon and transgene (GFP) expression in the cytosol was assessed by Western blot. Data are representative of two independent experiments. THP‐1s expressing either a control guide RNA or targeting cGAS and STING were generated and stimulated with 1,000 U/ml IFN‐α for 4 h and protein levels assessed by Western blot. THP‐1s deficient in cGAS and STING were stimulated with AdV (50,000 pp/cell) and 20 μg/ml h9C12, 200 ng/well HT‐DNA, 10 μM Nigericin or 10 ng/ml LPS for 16 h. Data show combined data (mean ± s.e.m) of three experiments with absolute protein values (upper panel) or as % cytokine output of KO cells relative to Ctrl‐treated cells (lower panel), * P ≤ 0.05, *** P ≤ 0.001 unpaired, two‐tailed t ‐test). WT THP‐1s were treated with 5 μM H151 for 30 min before stimulation as in (D). Data in upper panel are representative of three independent experiments. Data in lower panel show combined data of these three experiments (mean ± s.e.m) showing H151‐treated cells relative to media treated cells (* P ≤ 0.05, *** P ≤ 0.001 unpaired, two‐tailed t ‐test). ASC‐GFP THP‐1s were treated with 5 μM H151 for 30 min before stimulation with AdV‐mCherry (50,000 pp/cell) + 20 μg/ml h9C12 or 20 mg/ml IVIg or 200 ng/ well HT‐DNA for 8 h. A representative image (scale bar 50 μm) and quantification of number of cells with ASC specks from one representative experiment of three are shown. Source data are available online for this figure.

    Article Snippet: Nigericin sodium salt was purchased from Enzo Life Sciences (Catalogue no: BML‐CA421‐0005) and was reconstituted at 10 mM in ethanol.

    Techniques: Activation Assay, Derivative Assay, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Generated, Two Tailed Test

    IL‐1β and TNF release in response to AdV and h9C12 monoclonal antibody is TRIM21 dependent Schematic showing the h9C12 antibody mutants that no longer engage Trim21 (H433A) or FC receptors (LLAA). (B) Primed or (C) un‐primed HMDM were stimulated with AdV (50,000 pp/cell) +/− h9C12 (20 μg/ml) for 16 h ( n = 4, mean ± s.e.m. * P ≤ 0.05, paired, two‐tailed t‐ test). Ctrl or Trim21‐deficient THP‐1s were stimulated for 4 h with 1,000 U/ml IFNα and TRIM21 expression measured by immunoblot. WT or TRIM21‐deficient THP‐1s were stimulated with AdV (50,000 pp/cell) and 20 μg/ml h9C12 or 20 mg/ml IVIg or with 200 ng/well HT‐DNA or 10 μM Nigericin for 16 h, and IL‐1β (E) and TNF (F) measured in the supernatant by ELISA ( n = 4 (E) or n = 3 (F), mean ± s.e.m. * P ≤ 0.05, ** P ≤ 0.005, *** P ≤ 0.001 unpaired, two‐tailed t ‐test). WT mice were injected i.v. with 2.5 μg WT or H433A h9C12 antibody and then the next day injected i.v. with 2.5 × 10 11 pp AdV‐GFP. 4 h later spleens were harvested and neutrophil influx measured by flow cytometry ( n = 6 mean ± s.e.m. *** P ≤ 0.001 unpaired, two‐tailed t ‐test). HMDM were stimulated with AdV‐GFP (250 pp/cell) in the presence of h9C12 antibodies at indicated doses. Infection was measured after 24 h by flow cytometry. Infection relative to AdV alone is shown graphed on the left ( n = 4 mean ± s.e.m). Representative plots and a comparison of the different h9C12 mutants at 0.2 μg/ml are also shown in the graph on the right ( n = 4, mean ± s.e.m.). Source data are available online for this figure.

    Journal: The EMBO Journal

    Article Title: Antibody and DNA sensing pathways converge to activate the inflammasome during primary human macrophage infection

    doi: 10.15252/embj.2018101365

    Figure Lengend Snippet: IL‐1β and TNF release in response to AdV and h9C12 monoclonal antibody is TRIM21 dependent Schematic showing the h9C12 antibody mutants that no longer engage Trim21 (H433A) or FC receptors (LLAA). (B) Primed or (C) un‐primed HMDM were stimulated with AdV (50,000 pp/cell) +/− h9C12 (20 μg/ml) for 16 h ( n = 4, mean ± s.e.m. * P ≤ 0.05, paired, two‐tailed t‐ test). Ctrl or Trim21‐deficient THP‐1s were stimulated for 4 h with 1,000 U/ml IFNα and TRIM21 expression measured by immunoblot. WT or TRIM21‐deficient THP‐1s were stimulated with AdV (50,000 pp/cell) and 20 μg/ml h9C12 or 20 mg/ml IVIg or with 200 ng/well HT‐DNA or 10 μM Nigericin for 16 h, and IL‐1β (E) and TNF (F) measured in the supernatant by ELISA ( n = 4 (E) or n = 3 (F), mean ± s.e.m. * P ≤ 0.05, ** P ≤ 0.005, *** P ≤ 0.001 unpaired, two‐tailed t ‐test). WT mice were injected i.v. with 2.5 μg WT or H433A h9C12 antibody and then the next day injected i.v. with 2.5 × 10 11 pp AdV‐GFP. 4 h later spleens were harvested and neutrophil influx measured by flow cytometry ( n = 6 mean ± s.e.m. *** P ≤ 0.001 unpaired, two‐tailed t ‐test). HMDM were stimulated with AdV‐GFP (250 pp/cell) in the presence of h9C12 antibodies at indicated doses. Infection was measured after 24 h by flow cytometry. Infection relative to AdV alone is shown graphed on the left ( n = 4 mean ± s.e.m). Representative plots and a comparison of the different h9C12 mutants at 0.2 μg/ml are also shown in the graph on the right ( n = 4, mean ± s.e.m.). Source data are available online for this figure.

    Article Snippet: Nigericin sodium salt was purchased from Enzo Life Sciences (Catalogue no: BML‐CA421‐0005) and was reconstituted at 10 mM in ethanol.

    Techniques: Two Tailed Test, Expressing, Enzyme-linked Immunosorbent Assay, Mouse Assay, Injection, Flow Cytometry, Cytometry, Infection

    TRIM21 is involved in inflammasome activation rather than enhancing pro‐IL‐1β expression HMDM were stimulated with AdV (50,000 pp/cell) and antibody (h9C12; 20 μg/ml, IVIg: 20 mg/ml) or 10 μg/ml pI:C for 3 h and gene expression was measured by qPCR ( n = 4 mean ± s.e.m). HMDM were stimulated as in (A), or with 10 ng/ml LPS for 6 h and pro‐IL‐1β levels in the cytosol measured by Western blot. Blot is representative of three independent donors. HMDM were stimulated as in (A), or with 10 ng/ml LPS for 3 h and NLRP3 mRNA expression measured by qPCR. Data show average ± s.d. of two independent donors. HMDM were stimulated as in (B) and NLRP3 levels in the cytosol measured by Western blot. Blot is representative of two independent donors. TLR9 mRNA levels from PBMCs, CD19 +ve B cells, CD14 +ve monocytes or HMDM derived from CD14 +ve monocytes were assessed by qPCR, and copy number was determined relative to actin copy number ( n = 5 mean ± s.e.m). THP‐1s expressing ASC‐GFP were stimulated for 6 h with virus and antibody or 200 ng/well HT‐DNA or 10 μM Nigericin, in the presence of the pan‐caspase inhibitor zVAD‐fmk. A representative image (scale bar 100 μm) and quantification of number of cells with ASC specks from three independent experiments (mean ± s.e.m, * P ≤ 0.05, paired, two‐tailed t‐ test) are shown. Source data are available online for this figure.

    Journal: The EMBO Journal

    Article Title: Antibody and DNA sensing pathways converge to activate the inflammasome during primary human macrophage infection

    doi: 10.15252/embj.2018101365

    Figure Lengend Snippet: TRIM21 is involved in inflammasome activation rather than enhancing pro‐IL‐1β expression HMDM were stimulated with AdV (50,000 pp/cell) and antibody (h9C12; 20 μg/ml, IVIg: 20 mg/ml) or 10 μg/ml pI:C for 3 h and gene expression was measured by qPCR ( n = 4 mean ± s.e.m). HMDM were stimulated as in (A), or with 10 ng/ml LPS for 6 h and pro‐IL‐1β levels in the cytosol measured by Western blot. Blot is representative of three independent donors. HMDM were stimulated as in (A), or with 10 ng/ml LPS for 3 h and NLRP3 mRNA expression measured by qPCR. Data show average ± s.d. of two independent donors. HMDM were stimulated as in (B) and NLRP3 levels in the cytosol measured by Western blot. Blot is representative of two independent donors. TLR9 mRNA levels from PBMCs, CD19 +ve B cells, CD14 +ve monocytes or HMDM derived from CD14 +ve monocytes were assessed by qPCR, and copy number was determined relative to actin copy number ( n = 5 mean ± s.e.m). THP‐1s expressing ASC‐GFP were stimulated for 6 h with virus and antibody or 200 ng/well HT‐DNA or 10 μM Nigericin, in the presence of the pan‐caspase inhibitor zVAD‐fmk. A representative image (scale bar 100 μm) and quantification of number of cells with ASC specks from three independent experiments (mean ± s.e.m, * P ≤ 0.05, paired, two‐tailed t‐ test) are shown. Source data are available online for this figure.

    Article Snippet: Nigericin sodium salt was purchased from Enzo Life Sciences (Catalogue no: BML‐CA421‐0005) and was reconstituted at 10 mM in ethanol.

    Techniques: Activation Assay, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Derivative Assay, Two Tailed Test

    NLRP3 is required for inflammasome activation downstream of TRIM21 Primed HMDM were treated with MCC950 (1 μM) for 30 min before stimulation overnight with AdV (50,000 pp/cell) and h9C12 (20 μg/ml) or IVIg (20 mg/ml) or 10 μM Nigericin. IL‐1β (A) or TNF (B) or LDH release (C) was measured in cell supernatants. Data show average ± s.d. of two independent donors and are representative of three independent experiments. WT, NLRP3, ASC or caspase‐1‐deficient THP‐1s were stimulated as in (A) and IL‐1β in the supernatant measured by ELISA. Data are representative of two independent experiments. HMDM were primed with 10 μg/ml pI:C for 2 h, treated with 100 μM VX‐765 for 30 min before stimulation for 16 h as in (A). IL‐1β or LDH release was measured in cell supernatants. Data show average ± s.d. of two independent donors. Primed HMDM were treated with KCl as indicated for 1 h, before being stimulated for a further 6 h as in (A). IL‐1β and TNF were measured in cell supernatants by ELISA. Data are representative of three independent donors. Primed HMDM were stimulated with AdV and 20 μg/ml h9C12 or 10 μM Nigericin for 3 h and then immunostained for ASC or intracellular antibody. Co‐localisation was assessed by confocal microscopy. Data are representative of two independent donors.

    Journal: The EMBO Journal

    Article Title: Antibody and DNA sensing pathways converge to activate the inflammasome during primary human macrophage infection

    doi: 10.15252/embj.2018101365

    Figure Lengend Snippet: NLRP3 is required for inflammasome activation downstream of TRIM21 Primed HMDM were treated with MCC950 (1 μM) for 30 min before stimulation overnight with AdV (50,000 pp/cell) and h9C12 (20 μg/ml) or IVIg (20 mg/ml) or 10 μM Nigericin. IL‐1β (A) or TNF (B) or LDH release (C) was measured in cell supernatants. Data show average ± s.d. of two independent donors and are representative of three independent experiments. WT, NLRP3, ASC or caspase‐1‐deficient THP‐1s were stimulated as in (A) and IL‐1β in the supernatant measured by ELISA. Data are representative of two independent experiments. HMDM were primed with 10 μg/ml pI:C for 2 h, treated with 100 μM VX‐765 for 30 min before stimulation for 16 h as in (A). IL‐1β or LDH release was measured in cell supernatants. Data show average ± s.d. of two independent donors. Primed HMDM were treated with KCl as indicated for 1 h, before being stimulated for a further 6 h as in (A). IL‐1β and TNF were measured in cell supernatants by ELISA. Data are representative of three independent donors. Primed HMDM were stimulated with AdV and 20 μg/ml h9C12 or 10 μM Nigericin for 3 h and then immunostained for ASC or intracellular antibody. Co‐localisation was assessed by confocal microscopy. Data are representative of two independent donors.

    Article Snippet: Nigericin sodium salt was purchased from Enzo Life Sciences (Catalogue no: BML‐CA421‐0005) and was reconstituted at 10 mM in ethanol.

    Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Confocal Microscopy

    Inhibition of AR prevented –HG-induced NLRP3 inflammasome–mediated release of innate immune cytokines in Thp1 cells. Thp1 cells were preincubated (4 hours) with HG (25 mM) and then incubated with fidarestat (10 μΜ) for 30 minutes, followed by ATP (2 mM) for 1 hour. (a) Equal amount of proteins in the cell extracts (CEs) were subjected to Western blot analysis to determine the expression of inflammasome complex proteins NLRP3, ASC, caspase-1, and innate immune cytokines IL-1 β and IL-18, using specific antibodies. Equal amounts of proteins in the lyophilized culture media (CM) were also subjected to Western blot analysis for IL-1 β and IL-18. (b) IL-1 β release in cell culture supernatant was also determined by a specific ELISA kit. (c) IL-1 β mRNA levels in Thp1 cells were determined by reverse transcription-PCR, as described in Methods. (d) Thp1 cells were incubated (4 hours) with HG (25 mM) and then incubated with fidarestat (10 μM) for 30 minutes, followed by ATP (2 mM) and nigericin (20 µM) for 1 hour without or with addition of KCl (130 mM). Equal amounts of proteins in the lyophilized CM andCEs were subjected to Western blot analysis for IL-1 β . The representative cropped blots are shown. The bars represent the mean ± standard error of the mean (n ≥ 4). Fid, fidarestat.

    Journal: Endocrinology

    Article Title: Aldose Reductase Mediates NLRP3 Inflammasome–Initiated Innate Immune Response in Hyperglycemia-Induced Thp1 Monocytes and Male Mice

    doi: 10.1210/en.2017-00294

    Figure Lengend Snippet: Inhibition of AR prevented –HG-induced NLRP3 inflammasome–mediated release of innate immune cytokines in Thp1 cells. Thp1 cells were preincubated (4 hours) with HG (25 mM) and then incubated with fidarestat (10 μΜ) for 30 minutes, followed by ATP (2 mM) for 1 hour. (a) Equal amount of proteins in the cell extracts (CEs) were subjected to Western blot analysis to determine the expression of inflammasome complex proteins NLRP3, ASC, caspase-1, and innate immune cytokines IL-1 β and IL-18, using specific antibodies. Equal amounts of proteins in the lyophilized culture media (CM) were also subjected to Western blot analysis for IL-1 β and IL-18. (b) IL-1 β release in cell culture supernatant was also determined by a specific ELISA kit. (c) IL-1 β mRNA levels in Thp1 cells were determined by reverse transcription-PCR, as described in Methods. (d) Thp1 cells were incubated (4 hours) with HG (25 mM) and then incubated with fidarestat (10 μM) for 30 minutes, followed by ATP (2 mM) and nigericin (20 µM) for 1 hour without or with addition of KCl (130 mM). Equal amounts of proteins in the lyophilized CM andCEs were subjected to Western blot analysis for IL-1 β . The representative cropped blots are shown. The bars represent the mean ± standard error of the mean (n ≥ 4). Fid, fidarestat.

    Article Snippet: IL-1 β , IL-18, cryopyrin and ASC antibodies, and nigericin were obtained from Santa Cruz Biotechnology, as was radioimmunoprecipitation assay (RIPA) buffer.

    Techniques: Inhibition, Incubation, Western Blot, Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay, Polymerase Chain Reaction

    Febuxostat restored intracellular ATP by activating salvage pathway. ( a ) Schema of purine metabolism. ( b ) Primed BMDMs were pretreated 30 min with vehicle or febuxostat, and then stimulated 90 min with nigericin. Intracellular ATP, ADP and AMP were measured by HPLC method. Total adenylate was calculated. Intracellular uric acid level was measured. Data are representative of two independent experiments in which the same data were obtained and shown as mean ± SD. * p

    Journal: Scientific Reports

    Article Title: Febuxostat, a Xanthine Oxidoreductase Inhibitor, Decreases NLRP3-dependent Inflammation in Macrophages by Activating the Purine Salvage Pathway and Restoring Cellular Bioenergetics

    doi: 10.1038/s41598-019-53965-x

    Figure Lengend Snippet: Febuxostat restored intracellular ATP by activating salvage pathway. ( a ) Schema of purine metabolism. ( b ) Primed BMDMs were pretreated 30 min with vehicle or febuxostat, and then stimulated 90 min with nigericin. Intracellular ATP, ADP and AMP were measured by HPLC method. Total adenylate was calculated. Intracellular uric acid level was measured. Data are representative of two independent experiments in which the same data were obtained and shown as mean ± SD. * p

    Article Snippet: The primed cells were incubated 30 min with vehicle, febuxostat (200 μM, Teijin Pharma Ltd., Tokyo, Japan) or MitoTEMPO (500 μM, ENZO Life Sciences, New York, NY) in incomplete medium (without FBS), and then stimulated with nigericin (2.5 μM, AppliChem GmbH, Germany or Sigma-Aldrich) or MSU crystal (0.25 mg/mL) for the indicated time.

    Techniques: High Performance Liquid Chromatography

    Febuxostat inhibits cell death in mitochondrial ROS-independent manner. ( a ) Primed BMDMs were pretreated 30 min with vehicle, febuxostat or MitoTEMPO, and then stimulated 2 h with nigericin. LDH activity in the supernatant was measured. ( b ) Primed BMDMs were pretreated 30 min with vehicle, febuxostat or MitoTEMPO, and then stimulated 6 h with nigericin. After stimulation, cells were stained with calcein for living cell and PI for dead cell. Data are representative of three independent experiments performed in triplicate and shown as mean ± SD. ### p

    Journal: Scientific Reports

    Article Title: Febuxostat, a Xanthine Oxidoreductase Inhibitor, Decreases NLRP3-dependent Inflammation in Macrophages by Activating the Purine Salvage Pathway and Restoring Cellular Bioenergetics

    doi: 10.1038/s41598-019-53965-x

    Figure Lengend Snippet: Febuxostat inhibits cell death in mitochondrial ROS-independent manner. ( a ) Primed BMDMs were pretreated 30 min with vehicle, febuxostat or MitoTEMPO, and then stimulated 2 h with nigericin. LDH activity in the supernatant was measured. ( b ) Primed BMDMs were pretreated 30 min with vehicle, febuxostat or MitoTEMPO, and then stimulated 6 h with nigericin. After stimulation, cells were stained with calcein for living cell and PI for dead cell. Data are representative of three independent experiments performed in triplicate and shown as mean ± SD. ### p

    Article Snippet: The primed cells were incubated 30 min with vehicle, febuxostat (200 μM, Teijin Pharma Ltd., Tokyo, Japan) or MitoTEMPO (500 μM, ENZO Life Sciences, New York, NY) in incomplete medium (without FBS), and then stimulated with nigericin (2.5 μM, AppliChem GmbH, Germany or Sigma-Aldrich) or MSU crystal (0.25 mg/mL) for the indicated time.

    Techniques: Activity Assay, Staining

    Restored intracellular ATP is involved in the inhibitory effects of febuxostat. ( a ) Primed BMDMs were pretreated 30 min with vehicle, febuxostat or MitoTEMPO, and then stimulated 2 h with nigericin. Intracellular ATP was measured by luminescence method. ( b ) Primed BMDMs were pretreated 30 min with vehicle, febuxostat or MitoTEMPO, and then stimulated 90 min with nigericin. After stimulation, cells were loaded with TMRE. Data are representative of three independent experiments performed in triplicate and shown as mean ± SD. # p

    Journal: Scientific Reports

    Article Title: Febuxostat, a Xanthine Oxidoreductase Inhibitor, Decreases NLRP3-dependent Inflammation in Macrophages by Activating the Purine Salvage Pathway and Restoring Cellular Bioenergetics

    doi: 10.1038/s41598-019-53965-x

    Figure Lengend Snippet: Restored intracellular ATP is involved in the inhibitory effects of febuxostat. ( a ) Primed BMDMs were pretreated 30 min with vehicle, febuxostat or MitoTEMPO, and then stimulated 2 h with nigericin. Intracellular ATP was measured by luminescence method. ( b ) Primed BMDMs were pretreated 30 min with vehicle, febuxostat or MitoTEMPO, and then stimulated 90 min with nigericin. After stimulation, cells were loaded with TMRE. Data are representative of three independent experiments performed in triplicate and shown as mean ± SD. # p

    Article Snippet: The primed cells were incubated 30 min with vehicle, febuxostat (200 μM, Teijin Pharma Ltd., Tokyo, Japan) or MitoTEMPO (500 μM, ENZO Life Sciences, New York, NY) in incomplete medium (without FBS), and then stimulated with nigericin (2.5 μM, AppliChem GmbH, Germany or Sigma-Aldrich) or MSU crystal (0.25 mg/mL) for the indicated time.

    Techniques:

    Febuxostat improves cellular bioenergetics. ( a ) Heatmap image of metabolome data. Primed BMDMs were pretreated 30 min with vehicle or febuxostat, and then stimulated 90 min with nigericin. Cell extracts were used for metabolome analysis. Red indicates increased expression whereas green indicates decreased expression. ( b ) Illustration of mitochondrial respiratory capacity. ( c,d ) Primed BMDMs were pretreated 30 min with vehicle, febuxostat or MitoTEMPO, and then stimulated 90 min with nigericin. OCR was measured with the Seahorse XFp Extracellular Flux analyser. Basal respiration and ATP production were calculated by subtracting OCR value at 73.7 and at 34.3 min from OCR value at 14.6 min in ( c ), respectively. Data are shown as mean ± SD of four independent experiments in singlet. * p

    Journal: Scientific Reports

    Article Title: Febuxostat, a Xanthine Oxidoreductase Inhibitor, Decreases NLRP3-dependent Inflammation in Macrophages by Activating the Purine Salvage Pathway and Restoring Cellular Bioenergetics

    doi: 10.1038/s41598-019-53965-x

    Figure Lengend Snippet: Febuxostat improves cellular bioenergetics. ( a ) Heatmap image of metabolome data. Primed BMDMs were pretreated 30 min with vehicle or febuxostat, and then stimulated 90 min with nigericin. Cell extracts were used for metabolome analysis. Red indicates increased expression whereas green indicates decreased expression. ( b ) Illustration of mitochondrial respiratory capacity. ( c,d ) Primed BMDMs were pretreated 30 min with vehicle, febuxostat or MitoTEMPO, and then stimulated 90 min with nigericin. OCR was measured with the Seahorse XFp Extracellular Flux analyser. Basal respiration and ATP production were calculated by subtracting OCR value at 73.7 and at 34.3 min from OCR value at 14.6 min in ( c ), respectively. Data are shown as mean ± SD of four independent experiments in singlet. * p

    Article Snippet: The primed cells were incubated 30 min with vehicle, febuxostat (200 μM, Teijin Pharma Ltd., Tokyo, Japan) or MitoTEMPO (500 μM, ENZO Life Sciences, New York, NY) in incomplete medium (without FBS), and then stimulated with nigericin (2.5 μM, AppliChem GmbH, Germany or Sigma-Aldrich) or MSU crystal (0.25 mg/mL) for the indicated time.

    Techniques: Expressing

    Febuxostat inhibits IL-1β secretion in mitochondrial ROS-independent and -dependent manners. ( a ) Primed BMDMs were pretreated 30 min with vehicle or febuxostat, and then stimulated 2 h with nigericin or MSU. Supernatant and cell lysate were used for immunoblotting. Data are representative of two independent experiments in which the same data were obtained. ( b ) Primed BMDMs were pretreated 30 min with vehicle, febuxostat or MitoTEMPO, and then stimulated 2 h with nigericin or MSU. IL-1β in the supernatant was analysed by ELISA. ( c ) Primed BMDMs were pretreated 30 min with vehicle, febuxostat or MitoTEMPO, and then stimulated 90 min with nigericin or MSU. After stimulation, cells were loaded with DHR123. Data are representative of three independent experiments performed in triplicate and shown as mean ± SD. # p

    Journal: Scientific Reports

    Article Title: Febuxostat, a Xanthine Oxidoreductase Inhibitor, Decreases NLRP3-dependent Inflammation in Macrophages by Activating the Purine Salvage Pathway and Restoring Cellular Bioenergetics

    doi: 10.1038/s41598-019-53965-x

    Figure Lengend Snippet: Febuxostat inhibits IL-1β secretion in mitochondrial ROS-independent and -dependent manners. ( a ) Primed BMDMs were pretreated 30 min with vehicle or febuxostat, and then stimulated 2 h with nigericin or MSU. Supernatant and cell lysate were used for immunoblotting. Data are representative of two independent experiments in which the same data were obtained. ( b ) Primed BMDMs were pretreated 30 min with vehicle, febuxostat or MitoTEMPO, and then stimulated 2 h with nigericin or MSU. IL-1β in the supernatant was analysed by ELISA. ( c ) Primed BMDMs were pretreated 30 min with vehicle, febuxostat or MitoTEMPO, and then stimulated 90 min with nigericin or MSU. After stimulation, cells were loaded with DHR123. Data are representative of three independent experiments performed in triplicate and shown as mean ± SD. # p

    Article Snippet: The primed cells were incubated 30 min with vehicle, febuxostat (200 μM, Teijin Pharma Ltd., Tokyo, Japan) or MitoTEMPO (500 μM, ENZO Life Sciences, New York, NY) in incomplete medium (without FBS), and then stimulated with nigericin (2.5 μM, AppliChem GmbH, Germany or Sigma-Aldrich) or MSU crystal (0.25 mg/mL) for the indicated time.

    Techniques: Enzyme-linked Immunosorbent Assay

    Schema of the mechanisms underlying the inhibitory effect of febuxostat on NLRP3 inflammasome activation. ( a ) MSU mainly activates mitoROS-dependent pathway, while nigericin mainly activates an iATP loss-dependent pathway (Black arrows in bold). In particular, nigericin induces mitochondrial dysfunction, and iATP loss by the disturbance of oxidative phosphorylation, finally leading to IL-1β secretion and cell death. Concomitantly, iATP degrades into uric acid via purine degradation pathway. Red arrows indicate the effects of NLRP3 inflammasome activators. ( b ) In the case of stimuli with mitoROS production, such as MSU crystals, febuxostat inhibits IL-1β secretion through the inhibition of mitoROS production. In the case of nigericin stimulation, febuxostat inhibits IL-1β secretion by restoring iATP and improving mitochondrial function via the salvage pathway activation. Blue arrows indicate the effects of febuxostat on NLRP3 inflammasome activation pathways.

    Journal: Scientific Reports

    Article Title: Febuxostat, a Xanthine Oxidoreductase Inhibitor, Decreases NLRP3-dependent Inflammation in Macrophages by Activating the Purine Salvage Pathway and Restoring Cellular Bioenergetics

    doi: 10.1038/s41598-019-53965-x

    Figure Lengend Snippet: Schema of the mechanisms underlying the inhibitory effect of febuxostat on NLRP3 inflammasome activation. ( a ) MSU mainly activates mitoROS-dependent pathway, while nigericin mainly activates an iATP loss-dependent pathway (Black arrows in bold). In particular, nigericin induces mitochondrial dysfunction, and iATP loss by the disturbance of oxidative phosphorylation, finally leading to IL-1β secretion and cell death. Concomitantly, iATP degrades into uric acid via purine degradation pathway. Red arrows indicate the effects of NLRP3 inflammasome activators. ( b ) In the case of stimuli with mitoROS production, such as MSU crystals, febuxostat inhibits IL-1β secretion through the inhibition of mitoROS production. In the case of nigericin stimulation, febuxostat inhibits IL-1β secretion by restoring iATP and improving mitochondrial function via the salvage pathway activation. Blue arrows indicate the effects of febuxostat on NLRP3 inflammasome activation pathways.

    Article Snippet: The primed cells were incubated 30 min with vehicle, febuxostat (200 μM, Teijin Pharma Ltd., Tokyo, Japan) or MitoTEMPO (500 μM, ENZO Life Sciences, New York, NY) in incomplete medium (without FBS), and then stimulated with nigericin (2.5 μM, AppliChem GmbH, Germany or Sigma-Aldrich) or MSU crystal (0.25 mg/mL) for the indicated time.

    Techniques: Activation Assay, Inhibition

    A model integrating planarian bioelectrics to regenerative outcomes. ( A ) Cells at the surfaces of wound blastema (inserts) are predicted to measure the difference between their own depolarization (V mem (B)) and the average depolarization (V mem (Int)) of neighboring cells just interior to the wound blastema. If this difference is larger than some threshold value, the brain-head pathway is activated; if the difference is smaller than this threshold or negative, the tail pathway is activated. Branching between pathways is modeled by logistic-function kinetics. Local mutual inhibition by Notum and β -catenin is active in otherwise-untreated WT animals. ( B ) Treatment with nigericin solution (symbolized by orange lightning bolt arrow ) immediately after amputation depolarizes both wound blastema, leading to brain-head pathway activation and head regeneration at both wounds. Excessive blastema depolarization turns local mutual inhibition by Notum and β -catenin off. To see this figure in color, go online.

    Journal: Biophysical Journal

    Article Title: The Role of Early Bioelectric Signals in the Regeneration of Planarian Anterior/Posterior Polarity

    doi: 10.1016/j.bpj.2019.01.029

    Figure Lengend Snippet: A model integrating planarian bioelectrics to regenerative outcomes. ( A ) Cells at the surfaces of wound blastema (inserts) are predicted to measure the difference between their own depolarization (V mem (B)) and the average depolarization (V mem (Int)) of neighboring cells just interior to the wound blastema. If this difference is larger than some threshold value, the brain-head pathway is activated; if the difference is smaller than this threshold or negative, the tail pathway is activated. Branching between pathways is modeled by logistic-function kinetics. Local mutual inhibition by Notum and β -catenin is active in otherwise-untreated WT animals. ( B ) Treatment with nigericin solution (symbolized by orange lightning bolt arrow ) immediately after amputation depolarizes both wound blastema, leading to brain-head pathway activation and head regeneration at both wounds. Excessive blastema depolarization turns local mutual inhibition by Notum and β -catenin off. To see this figure in color, go online.

    Article Snippet: Immediately after cutting, fragments were transferred to either a 0.24 μ M nigericin (Adipogen) + 15 mM potassium gluconate (Sigma-Aldrich, St. Louis, MO) solution (“nigericin solution”) or a 0.08 μ M Monensin (Cayman Chemical, Ann Arbor, MI) + 90 mM sodium gluconate (Sigma-Aldrich) solution (“monensin solution”).

    Techniques: Inhibition, Activation Assay

    A brief, 3-h depolarization changes early expression of notum . ( A ) DiBAC 4 (3) staining of PT fragments in controls treated for 3 h with ethanol control solutions is shown, imaged at ( a ) 3 h and ( b ) 6 h, focusing on relative intensity distributions at the anterior ( green arrow ) compared to posterior ( blue arrow ) blastema. This is compared to fragments treated for 3 h after amputation in 0.24 μ M nigericin + 15 mM potassium gluconate, imaged at ( c ) 3 h and ( d ) 6 h. ( B ) Quantification of the average DiBAC 4 (3) fluorescence intensity at the anterior blastema ( green dots ) and posterior blastema ( purple ) in the nigericin-treated fragments at 3 h ( p > 0.5, N = 19, paired t -test) and 6 h ( p

    Journal: Biophysical Journal

    Article Title: The Role of Early Bioelectric Signals in the Regeneration of Planarian Anterior/Posterior Polarity

    doi: 10.1016/j.bpj.2019.01.029

    Figure Lengend Snippet: A brief, 3-h depolarization changes early expression of notum . ( A ) DiBAC 4 (3) staining of PT fragments in controls treated for 3 h with ethanol control solutions is shown, imaged at ( a ) 3 h and ( b ) 6 h, focusing on relative intensity distributions at the anterior ( green arrow ) compared to posterior ( blue arrow ) blastema. This is compared to fragments treated for 3 h after amputation in 0.24 μ M nigericin + 15 mM potassium gluconate, imaged at ( c ) 3 h and ( d ) 6 h. ( B ) Quantification of the average DiBAC 4 (3) fluorescence intensity at the anterior blastema ( green dots ) and posterior blastema ( purple ) in the nigericin-treated fragments at 3 h ( p > 0.5, N = 19, paired t -test) and 6 h ( p

    Article Snippet: Immediately after cutting, fragments were transferred to either a 0.24 μ M nigericin (Adipogen) + 15 mM potassium gluconate (Sigma-Aldrich, St. Louis, MO) solution (“nigericin solution”) or a 0.08 μ M Monensin (Cayman Chemical, Ann Arbor, MI) + 90 mM sodium gluconate (Sigma-Aldrich) solution (“monensin solution”).

    Techniques: Expressing, Staining, Fluorescence

    β-catenin RNAi induces double-headed planaria without depolarization. ( A ) Schematic showing β-catenin -dsRNAi injection, which results in regeneration of double-headed planaria from cut fragments, is given. ( B ) ( a ) An example image of a β-catenin -dsRNAi-induced double-headed planarian is given, showing abnormal shapes and defects in remodeling toward a normal body shape during the course of regeneration, ( b ) compared to double-headed planaria induced by nigericin treatment and ( c ) one induced by combination of β-catenin dsRNAi and nigericin treatment. ( C ) DiBAC 4 (3) staining 3 h postamputation of a ( a ) WT fragment and ( b ) a fragment from a β-catenin -dsRNAi-injected animal amputated 1 week after injection is given, showing no relative difference in the V mem ( p > 0.05, paired t -test). ( c ) DiBAC 4 (3) staining of a fragment treated with nigericin is shown. ( D ) Quantification of the difference between DiBAC 4 (3) intensity of β-catenin -dsRNAi-injected fragments and their respective controls versus nigericin-treated fragments relative to their respective controls is shown. ∗∗ p

    Journal: Biophysical Journal

    Article Title: The Role of Early Bioelectric Signals in the Regeneration of Planarian Anterior/Posterior Polarity

    doi: 10.1016/j.bpj.2019.01.029

    Figure Lengend Snippet: β-catenin RNAi induces double-headed planaria without depolarization. ( A ) Schematic showing β-catenin -dsRNAi injection, which results in regeneration of double-headed planaria from cut fragments, is given. ( B ) ( a ) An example image of a β-catenin -dsRNAi-induced double-headed planarian is given, showing abnormal shapes and defects in remodeling toward a normal body shape during the course of regeneration, ( b ) compared to double-headed planaria induced by nigericin treatment and ( c ) one induced by combination of β-catenin dsRNAi and nigericin treatment. ( C ) DiBAC 4 (3) staining 3 h postamputation of a ( a ) WT fragment and ( b ) a fragment from a β-catenin -dsRNAi-injected animal amputated 1 week after injection is given, showing no relative difference in the V mem ( p > 0.05, paired t -test). ( c ) DiBAC 4 (3) staining of a fragment treated with nigericin is shown. ( D ) Quantification of the difference between DiBAC 4 (3) intensity of β-catenin -dsRNAi-injected fragments and their respective controls versus nigericin-treated fragments relative to their respective controls is shown. ∗∗ p

    Article Snippet: Immediately after cutting, fragments were transferred to either a 0.24 μ M nigericin (Adipogen) + 15 mM potassium gluconate (Sigma-Aldrich, St. Louis, MO) solution (“nigericin solution”) or a 0.08 μ M Monensin (Cayman Chemical, Ann Arbor, MI) + 90 mM sodium gluconate (Sigma-Aldrich) solution (“monensin solution”).

    Techniques: Injection, Staining

    Nigericin and monensin treatment depolarizes worm fragments and leads to regeneration of double-headed planaria. ( A ) Treatment timeline for ionophore (nigericin and monensin) solutions is shown. PT fragments were amputated from WT D. japonica and treated with 0.24 μ M nigericin + 15 mM potassium gluconate or with 0.08 μ M monensin + 90 mM sodium gluconate for the first 3 h postamputation side by side with corresponding ethanol in water controls. Animals were moved from treatment solutions into water and washed three times. Worms regenerated for 2 weeks before they were scored. ( B and C ) V mem reporter assay using DiBAC 4 (3) is shown. Brighter signal indicates relative depolarization, whereas lower intensity indicates relatively hyperpolarized cells. Green arrows indicate anterior blastema, and blue arrows indicate posterior blastema. ( B ) Treatment with nigericin solution is shown. ( a ) A DiBAC 4 (3)-stained control D. japonica PT fragment 3 h postamputation is shown. ( b and c ) Regenerative outcome of the control treatment, showing a single-headed worm, with a head at the anterior ( b ) and a tail at the posterior ( c ), is given. ( d ) A DiBAC 4 (3)-stained D. japonica PT fragment 3 h postamputation treated with nigericin solution is given, showing strong depolarization, ( e and f ) which results in 13% double-headed regenerative outcomes for worms, with a head both at the anterior ( e ) and the posterior ( f ) in significantly higher numbers than controls in which this phenotype was not observed ( p

    Journal: Biophysical Journal

    Article Title: The Role of Early Bioelectric Signals in the Regeneration of Planarian Anterior/Posterior Polarity

    doi: 10.1016/j.bpj.2019.01.029

    Figure Lengend Snippet: Nigericin and monensin treatment depolarizes worm fragments and leads to regeneration of double-headed planaria. ( A ) Treatment timeline for ionophore (nigericin and monensin) solutions is shown. PT fragments were amputated from WT D. japonica and treated with 0.24 μ M nigericin + 15 mM potassium gluconate or with 0.08 μ M monensin + 90 mM sodium gluconate for the first 3 h postamputation side by side with corresponding ethanol in water controls. Animals were moved from treatment solutions into water and washed three times. Worms regenerated for 2 weeks before they were scored. ( B and C ) V mem reporter assay using DiBAC 4 (3) is shown. Brighter signal indicates relative depolarization, whereas lower intensity indicates relatively hyperpolarized cells. Green arrows indicate anterior blastema, and blue arrows indicate posterior blastema. ( B ) Treatment with nigericin solution is shown. ( a ) A DiBAC 4 (3)-stained control D. japonica PT fragment 3 h postamputation is shown. ( b and c ) Regenerative outcome of the control treatment, showing a single-headed worm, with a head at the anterior ( b ) and a tail at the posterior ( c ), is given. ( d ) A DiBAC 4 (3)-stained D. japonica PT fragment 3 h postamputation treated with nigericin solution is given, showing strong depolarization, ( e and f ) which results in 13% double-headed regenerative outcomes for worms, with a head both at the anterior ( e ) and the posterior ( f ) in significantly higher numbers than controls in which this phenotype was not observed ( p

    Article Snippet: Immediately after cutting, fragments were transferred to either a 0.24 μ M nigericin (Adipogen) + 15 mM potassium gluconate (Sigma-Aldrich, St. Louis, MO) solution (“nigericin solution”) or a 0.08 μ M Monensin (Cayman Chemical, Ann Arbor, MI) + 90 mM sodium gluconate (Sigma-Aldrich) solution (“monensin solution”).

    Techniques: Reporter Assay, Staining