Structured Review

Enzo Biochem msu
Extracellular succinate signals via GPR91 to stimulate macrophages to release <t>IL-1β.</t> (A) GPR91 mRNA expression in WT (Janvier C57BL/6J) inflammatory BMDMs (M-CSF + IFN-γ) ± 100 ng/ml LPS, 500 µM succinate, or 10 ng/ml IL-1β for 24 h. n = 3 of Ct values. Succinate (Succ), IL-1β, and LPS related to basal (=1). Data are representative of three experiments. (B) Succinate levels (mass spectrophotometry area ratio) in medium from cultured BMDMs. Extracellular succinate from WT (littermates; black bars) and Sucnr1 −/− (gray bars), neutral (M, M-CSF), or inflammatory (M + IFN-γ) BMDMs ± 100 ng/ml LPS for 24 h is shown. n = 6 wells. Data are representative of three experiments. (C) IL-1β in supernatants of WT (Janvier C57BL/6J) and Sucnr1 −/− neutral or inflammatory BMDMs ± 100 ng/ml LPS for 24 h. n = 3 wells and are representative of seven experiments. (D) IL-1β mRNA levels from cell lysates from WT (Janvier C57BL/6J) or Sucnr1 −/− inflammatory BMDMs ± 100 ng/ml LPS at 4 h (related to WT basal = 1). n = 2–3 of Ct values. Data are representative of two experiments. (E) IL-1β levels measured in the supernatant of WT (Janvier C57BL/6J) and Sucnr1 −/− inflammatory BMDMs stimulated with 1 ng/ml LPS and 180 µg/ml <t>MSU.</t> n = 5–6 wells. Data are representative of two experiments. (F) Western blot of HIF-1α (representative blot of two experiments) and quantification (two experiments; 100% for no stimulus, WT, and Sucnr1 −/− ) at 6 h after stimulation with 500 µM succinate, 100 ng/ml LPS, or a combination of the two in WT littermate controls and Sucnr1 −/− inflammatory BMDMs. *, P
Msu, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 91/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "GPR91 senses extracellular succinate released from inflammatory macrophages and exacerbates rheumatoid arthritis"

Article Title: GPR91 senses extracellular succinate released from inflammatory macrophages and exacerbates rheumatoid arthritis

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20160061

Extracellular succinate signals via GPR91 to stimulate macrophages to release IL-1β. (A) GPR91 mRNA expression in WT (Janvier C57BL/6J) inflammatory BMDMs (M-CSF + IFN-γ) ± 100 ng/ml LPS, 500 µM succinate, or 10 ng/ml IL-1β for 24 h. n = 3 of Ct values. Succinate (Succ), IL-1β, and LPS related to basal (=1). Data are representative of three experiments. (B) Succinate levels (mass spectrophotometry area ratio) in medium from cultured BMDMs. Extracellular succinate from WT (littermates; black bars) and Sucnr1 −/− (gray bars), neutral (M, M-CSF), or inflammatory (M + IFN-γ) BMDMs ± 100 ng/ml LPS for 24 h is shown. n = 6 wells. Data are representative of three experiments. (C) IL-1β in supernatants of WT (Janvier C57BL/6J) and Sucnr1 −/− neutral or inflammatory BMDMs ± 100 ng/ml LPS for 24 h. n = 3 wells and are representative of seven experiments. (D) IL-1β mRNA levels from cell lysates from WT (Janvier C57BL/6J) or Sucnr1 −/− inflammatory BMDMs ± 100 ng/ml LPS at 4 h (related to WT basal = 1). n = 2–3 of Ct values. Data are representative of two experiments. (E) IL-1β levels measured in the supernatant of WT (Janvier C57BL/6J) and Sucnr1 −/− inflammatory BMDMs stimulated with 1 ng/ml LPS and 180 µg/ml MSU. n = 5–6 wells. Data are representative of two experiments. (F) Western blot of HIF-1α (representative blot of two experiments) and quantification (two experiments; 100% for no stimulus, WT, and Sucnr1 −/− ) at 6 h after stimulation with 500 µM succinate, 100 ng/ml LPS, or a combination of the two in WT littermate controls and Sucnr1 −/− inflammatory BMDMs. *, P
Figure Legend Snippet: Extracellular succinate signals via GPR91 to stimulate macrophages to release IL-1β. (A) GPR91 mRNA expression in WT (Janvier C57BL/6J) inflammatory BMDMs (M-CSF + IFN-γ) ± 100 ng/ml LPS, 500 µM succinate, or 10 ng/ml IL-1β for 24 h. n = 3 of Ct values. Succinate (Succ), IL-1β, and LPS related to basal (=1). Data are representative of three experiments. (B) Succinate levels (mass spectrophotometry area ratio) in medium from cultured BMDMs. Extracellular succinate from WT (littermates; black bars) and Sucnr1 −/− (gray bars), neutral (M, M-CSF), or inflammatory (M + IFN-γ) BMDMs ± 100 ng/ml LPS for 24 h is shown. n = 6 wells. Data are representative of three experiments. (C) IL-1β in supernatants of WT (Janvier C57BL/6J) and Sucnr1 −/− neutral or inflammatory BMDMs ± 100 ng/ml LPS for 24 h. n = 3 wells and are representative of seven experiments. (D) IL-1β mRNA levels from cell lysates from WT (Janvier C57BL/6J) or Sucnr1 −/− inflammatory BMDMs ± 100 ng/ml LPS at 4 h (related to WT basal = 1). n = 2–3 of Ct values. Data are representative of two experiments. (E) IL-1β levels measured in the supernatant of WT (Janvier C57BL/6J) and Sucnr1 −/− inflammatory BMDMs stimulated with 1 ng/ml LPS and 180 µg/ml MSU. n = 5–6 wells. Data are representative of two experiments. (F) Western blot of HIF-1α (representative blot of two experiments) and quantification (two experiments; 100% for no stimulus, WT, and Sucnr1 −/− ) at 6 h after stimulation with 500 µM succinate, 100 ng/ml LPS, or a combination of the two in WT littermate controls and Sucnr1 −/− inflammatory BMDMs. *, P

Techniques Used: Expressing, Spectrophotometry, Cell Culture, Western Blot

Related Articles

Transfection:

Article Title: Cyclic-di-GMP and cyclic-di-AMP activate the NLRP3 inflammasome
Article Snippet: .. THP-1 cells were primed with phorbol myristate acetate (PMA; 100 nM, over night; Sigma-Aldrich) and BMMs were primed with LPS (1 μg/ml, 3 h; E. coli ; type 025:86; Sigma-Aldrich) before stimulation with ATP (Sigma-Aldrich), MSU (Enzo Life Sciences, Farmingdale, NY), crystalline silica (Min-U-Sil 5, U.S. Silica, Berkeley Springs, WV), S. typhimurium (see online), L. pneumophila (see online; [ ]), or transfection with synthetic c-diGMP or c-diAMP ( > 99% pure; [ ]), cGMP (Enzo Life Sciences), GTP (Sigma-Aldrich) or poly(dA:dT) (Sigma-Aldrich), as indicated. .. Transfections were carried out with Lipofectamine 2000 (Invitrogen, Carlsbad, CA) or X-tremeGENE HP (Roche; Indianapolis, IN), respectively.

Cycling Probe Technology:

Article Title: NLRP1 promotes tumor growth by enhancing inflammasome activation and suppressing apoptosis in metastatic melanoma
Article Snippet: .. Caspase activity assay To determine the activity of the caspases of interest, cells were treated with DMSO as a vehicle control or an apoptosis inducer, AMD (5 μM) or CPT (1 μM) (BioVision, Milpitas, CA), for 18 h. To determine the effect of inflammasome activation on the caspase activities, cells were pre-treated with LT, MSU (50 μg/ml; Enzo Life Sciences), or recombinant human IL-1α (10 ng/ml; ThermoFisher Scientific) for 2 h prior to the addition of apoptosis inducers. .. Caspase-1 and -9 activities were measured using FAM-FLICA caspase assay kits (ImmunoChemistry Technologies, Bloomington, MN) with a fluorescence plate reader.

Activation Assay:

Article Title: NLRP1 promotes tumor growth by enhancing inflammasome activation and suppressing apoptosis in metastatic melanoma
Article Snippet: .. Caspase activity assay To determine the activity of the caspases of interest, cells were treated with DMSO as a vehicle control or an apoptosis inducer, AMD (5 μM) or CPT (1 μM) (BioVision, Milpitas, CA), for 18 h. To determine the effect of inflammasome activation on the caspase activities, cells were pre-treated with LT, MSU (50 μg/ml; Enzo Life Sciences), or recombinant human IL-1α (10 ng/ml; ThermoFisher Scientific) for 2 h prior to the addition of apoptosis inducers. .. Caspase-1 and -9 activities were measured using FAM-FLICA caspase assay kits (ImmunoChemistry Technologies, Bloomington, MN) with a fluorescence plate reader.

Synthesized:

Article Title: GPR91 senses extracellular succinate released from inflammatory macrophages and exacerbates rheumatoid arthritis
Article Snippet: .. In vitro stimulation of mouse macrophages or U937 cells U937 or mouse macrophages derived from 7–9-wk-old age-matched animals were incubated for up to 24 h in 96-well flat-bottom plates at a density of 105 cells per well in RPMI-1640 containing 1 or 100 ng/ml LPS (InvivoGen), 500 µM succinate (sodium succinate dibasic hexahydrate; Sigma-Aldrich), 10 ng/ml mouse IL-1β (PeproTech), 180 µg/ml MSU (Enzo Life Sciences), 5 µM GPR91 antagonist GPR91A1 (synthesized in house from ), and 10% human RA SFs (Asterand). .. After 24 h (or 7 h in the case of culture with 1 ng/ml LPS [2 h] followed by 180 µg/ml MSU [5 h]), cells were collected and processed for quantitative PCR as described in the next section.

Concentration Assay:

Article Title: Monosodium Urate Crystals Promote Innate Anti-Mycobacterial Immunity and Improve BCG Efficacy as a Vaccine against Tuberculosis
Article Snippet: .. After removal of extra-cellular bacilli, cells were stimulated or not with MSU (Enzo Life Sciences Inc.) at the concentration of 0.5, 5, 50 μg/ml and colony forming unit (CFU) assays were performed at day 3 and 5 post-infection, as previously described [ ]. .. In several experiments, any possible effects, induced by MSU on phagocytosis, were evaluated by CFU assay performed on dTHP-1 cells after 3-hour exposure with BCG, administrated at the MOI of 1 in the presence or absence of 0.05, 0.5, 5 μg/ml MSU.

Incubation:

Article Title: Inflammasome Activation by Cystine Crystals: Implications for the Pathogenesis of Cystinosis
Article Snippet: .. The next day, PBMCs were stimulated with 100 ng/ml LPS ( Escherichia coli serotype 055:B5; Sigma-Aldrich) for 2 hours, followed by incubation with cystine crystals or MSU (Alexis Biochemicals, San Diego, CA) for 6 hours. .. In some experiments, cells were preincubated 30 minutes before crystal addition with the caspase-1 inhibitor z-YVAD-fmk (Alexis Biochemicals), cytochalasin D (Sigma-Aldrich), cathepsin B inhibitor CA-074Me (Calbiochem), diphenyleneiodonium chloride (ENZO Life Sciences), or 130 mM KCl.

Article Title: GPR91 senses extracellular succinate released from inflammatory macrophages and exacerbates rheumatoid arthritis
Article Snippet: .. In vitro stimulation of mouse macrophages or U937 cells U937 or mouse macrophages derived from 7–9-wk-old age-matched animals were incubated for up to 24 h in 96-well flat-bottom plates at a density of 105 cells per well in RPMI-1640 containing 1 or 100 ng/ml LPS (InvivoGen), 500 µM succinate (sodium succinate dibasic hexahydrate; Sigma-Aldrich), 10 ng/ml mouse IL-1β (PeproTech), 180 µg/ml MSU (Enzo Life Sciences), 5 µM GPR91 antagonist GPR91A1 (synthesized in house from ), and 10% human RA SFs (Asterand). .. After 24 h (or 7 h in the case of culture with 1 ng/ml LPS [2 h] followed by 180 µg/ml MSU [5 h]), cells were collected and processed for quantitative PCR as described in the next section.

Activity Assay:

Article Title: NLRP1 promotes tumor growth by enhancing inflammasome activation and suppressing apoptosis in metastatic melanoma
Article Snippet: .. Caspase activity assay To determine the activity of the caspases of interest, cells were treated with DMSO as a vehicle control or an apoptosis inducer, AMD (5 μM) or CPT (1 μM) (BioVision, Milpitas, CA), for 18 h. To determine the effect of inflammasome activation on the caspase activities, cells were pre-treated with LT, MSU (50 μg/ml; Enzo Life Sciences), or recombinant human IL-1α (10 ng/ml; ThermoFisher Scientific) for 2 h prior to the addition of apoptosis inducers. .. Caspase-1 and -9 activities were measured using FAM-FLICA caspase assay kits (ImmunoChemistry Technologies, Bloomington, MN) with a fluorescence plate reader.

Caspase Activity Assay:

Article Title: NLRP1 promotes tumor growth by enhancing inflammasome activation and suppressing apoptosis in metastatic melanoma
Article Snippet: .. Caspase activity assay To determine the activity of the caspases of interest, cells were treated with DMSO as a vehicle control or an apoptosis inducer, AMD (5 μM) or CPT (1 μM) (BioVision, Milpitas, CA), for 18 h. To determine the effect of inflammasome activation on the caspase activities, cells were pre-treated with LT, MSU (50 μg/ml; Enzo Life Sciences), or recombinant human IL-1α (10 ng/ml; ThermoFisher Scientific) for 2 h prior to the addition of apoptosis inducers. .. Caspase-1 and -9 activities were measured using FAM-FLICA caspase assay kits (ImmunoChemistry Technologies, Bloomington, MN) with a fluorescence plate reader.

Recombinant:

Article Title: NLRP1 promotes tumor growth by enhancing inflammasome activation and suppressing apoptosis in metastatic melanoma
Article Snippet: .. Caspase activity assay To determine the activity of the caspases of interest, cells were treated with DMSO as a vehicle control or an apoptosis inducer, AMD (5 μM) or CPT (1 μM) (BioVision, Milpitas, CA), for 18 h. To determine the effect of inflammasome activation on the caspase activities, cells were pre-treated with LT, MSU (50 μg/ml; Enzo Life Sciences), or recombinant human IL-1α (10 ng/ml; ThermoFisher Scientific) for 2 h prior to the addition of apoptosis inducers. .. Caspase-1 and -9 activities were measured using FAM-FLICA caspase assay kits (ImmunoChemistry Technologies, Bloomington, MN) with a fluorescence plate reader.

Derivative Assay:

Article Title: GPR91 senses extracellular succinate released from inflammatory macrophages and exacerbates rheumatoid arthritis
Article Snippet: .. In vitro stimulation of mouse macrophages or U937 cells U937 or mouse macrophages derived from 7–9-wk-old age-matched animals were incubated for up to 24 h in 96-well flat-bottom plates at a density of 105 cells per well in RPMI-1640 containing 1 or 100 ng/ml LPS (InvivoGen), 500 µM succinate (sodium succinate dibasic hexahydrate; Sigma-Aldrich), 10 ng/ml mouse IL-1β (PeproTech), 180 µg/ml MSU (Enzo Life Sciences), 5 µM GPR91 antagonist GPR91A1 (synthesized in house from ), and 10% human RA SFs (Asterand). .. After 24 h (or 7 h in the case of culture with 1 ng/ml LPS [2 h] followed by 180 µg/ml MSU [5 h]), cells were collected and processed for quantitative PCR as described in the next section.

In Vitro:

Article Title: GPR91 senses extracellular succinate released from inflammatory macrophages and exacerbates rheumatoid arthritis
Article Snippet: .. In vitro stimulation of mouse macrophages or U937 cells U937 or mouse macrophages derived from 7–9-wk-old age-matched animals were incubated for up to 24 h in 96-well flat-bottom plates at a density of 105 cells per well in RPMI-1640 containing 1 or 100 ng/ml LPS (InvivoGen), 500 µM succinate (sodium succinate dibasic hexahydrate; Sigma-Aldrich), 10 ng/ml mouse IL-1β (PeproTech), 180 µg/ml MSU (Enzo Life Sciences), 5 µM GPR91 antagonist GPR91A1 (synthesized in house from ), and 10% human RA SFs (Asterand). .. After 24 h (or 7 h in the case of culture with 1 ng/ml LPS [2 h] followed by 180 µg/ml MSU [5 h]), cells were collected and processed for quantitative PCR as described in the next section.

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    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 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/nigericin/product/Enzo Biochem
    Average 92 stars, based on 3 article reviews
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    nigericin - by Bioz Stars, 2020-09
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    92
    Enzo Biochem flagellin
    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
    Flagellin, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 92/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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