mouse flag tagged nlrp3  (Addgene inc)

Journal: Journal of Cellular and Molecular Medicine

Article Title: Anti‐inflammatory effect of Trichospira verticillata via suppression of the NLRP3 inflammasome in neutrophilic asthma

doi: 10.1111/jcmm.18356

Figure Lengend Snippet: TVE specifically suppresses NLRP3 inflammasome. (A) Schematic outline of the NLRP3 inflammasome activation experiments in vitro. (B–C) PMA (500 nM)‐differentiated THP‐1 cells (B) and J774A.1 cells (C) were primed by LPS (100 ng/mL) for 3 h, before were treated with TVE for 2 h. Cell viability was analysed by EZ‐Cytox. (D–E) LPS‐primed THP‐1 cells were treated with TVE for 2 h and then stimulated for 1 h with nigericin (10 μM) (D) and ATP (5 mM) (E) with or without MCC950. (F–G) LPS‐primed J774A.1 cells were treated with TVE for 2 h and then stimulated for 30 min with nigericin (10 μM) (F), ATP (5 mM) (G) with or without MCC950. This data represents the investigation of the protein expression level of IL‐1β in supernatant, confirmed by immunoblot, using densitometry to assess intensity. (H‐J) IL‐1β (p17) and in the supernatants (Sup) and soluble lysates (Lys) were analysed by immunoblot. LPS‐primed J774A.1 cells were treated with TVE for 2 h and then stimulated for 3 h with MSU (100 μg/mL) (H) or dsDNA (2 μg/mL) (I) and flagellin (1.25 μg/mL) (J) by using Lipofectamine 3000™. One representative result of three independent experiments is shown. Values shown are reported as the means of technical triplicates ± SEM. One‐way ANOVA, Bonferroni post‐hoc test; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. ATP, Adenosine triphosphate; IL‐1β, Interleukin‐1beta; LPS, Lipopolysaccharide; MCC, MCC950; Nig, Nigericin; n.s., not significant; TVE, Trichospira verticillata (L.) S.F. Blake Extract.

Article Snippet: The purified mouse NLRP3 (BPS bioscience, 100189) (0.139 mg/mL) was incubated with TVE (50 μg/mL) or DMSO at 37°C for 30 min in the reaction buffer (100 mM Tris pH 7.8, 2.8 mM EDTA, 100 mM MgCl₂, 15 mM KCl, 655 mM NaCl).

Techniques: Activation Assay, In Vitro, Expressing, Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: Anti‐inflammatory effect of Trichospira verticillata via suppression of the NLRP3 inflammasome in neutrophilic asthma

doi: 10.1111/jcmm.18356

Figure Lengend Snippet: TVE suppresses NLRP3 inflammasome activation regardless of K + efflux, ROS and ATPase activity. (A) LPS‐primed J774A.1 cells were treated with TVE for 2 h and then stimulated with IMQ (200 μM) for 1 h. (B) LPS‐primed J774A.1 cells were treated with or without TVE or NAC for 2 h and then stimulated with ATP (5 mM) for 25 min. ROS levels are detected by a microplate reader using a DCFDA solution (20 μM). (C) The amount of NLRP3‐mediated ATP converted into ADP with TVE was determined by luminescence using the ADP‐Glo Assay. (D) and (E) The NLRP3‐Myc transfected‐HEK 293FT cells were treated with or without TVE for 2 h. The NLRP3‐NEK7 interaction was analysed by immunoprecipitation and immunoblot. One representative result of three independent experiments is shown. Values are shown reported as the means of technical triplicates ± SEM. One‐way ANOVA, Bonferroni post‐hoc test; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. LPS, Lipopolysaccharide; n.s., not significant; NAC, N‐acetyl‐l‐cysteine; ROS, reactive oxygen species; TVE, Trichospira verticillata (L.) S.F. Blake Extract.

Article Snippet: The purified mouse NLRP3 (BPS bioscience, 100189) (0.139 mg/mL) was incubated with TVE (50 μg/mL) or DMSO at 37°C for 30 min in the reaction buffer (100 mM Tris pH 7.8, 2.8 mM EDTA, 100 mM MgCl₂, 15 mM KCl, 655 mM NaCl).

Techniques: Activation Assay, Activity Assay, Glo Assay, Transfection, Immunoprecipitation, Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: Anti‐inflammatory effect of Trichospira verticillata via suppression of the NLRP3 inflammasome in neutrophilic asthma

doi: 10.1111/jcmm.18356

Figure Lengend Snippet: TVE blocks NLRP3 inflammasome assembly by hindering ASC oligomerization. (A) One representative ASC speck (indicated by arrow) images of at least 10 images are shown. LPS‐primed J774A.1 cells were treated with TVE for 2 h and then stimulated with ATP (5 mM) for 30 min. (B) LPS‐primed THP‐1 cells were treated with TVE for 2 h and then stimulated with nigericin (10 μM) for 30 min. ASC oligomerization in the cross‐linked cytosolic pellet was analysed by immunoblot. Scale bar, 20 μm. One representative result of three independent experiments is shown. Values are shown reported as the means of 10 replicates ± SEM. One‐way ANOVA, Bonferroni post‐hoc test; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. ASC, apoptosis‐associated speck‐like protein; LPS, Lipopolysaccharide; n.s., not significant; TVE, Trichospira verticillata (L.) S.F. Blake Extract.

Article Snippet: The purified mouse NLRP3 (BPS bioscience, 100189) (0.139 mg/mL) was incubated with TVE (50 μg/mL) or DMSO at 37°C for 30 min in the reaction buffer (100 mM Tris pH 7.8, 2.8 mM EDTA, 100 mM MgCl₂, 15 mM KCl, 655 mM NaCl).

Techniques: Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: Anti‐inflammatory effect of Trichospira verticillata via suppression of the NLRP3 inflammasome in neutrophilic asthma

doi: 10.1111/jcmm.18356

Figure Lengend Snippet: TVE inhibits NLRP3 inflammasome in lung epithelial cells. (A–B) A549 cells were treated with TVE for 2 h (A), or overnight (B). Cell viability was analysed by EZ‐CYTOX. (C–D) A549 cells and PMA (500 nM)‐differentiated THP‐1 cells were co‐cultured and primed by LPS (100 ng/mL) for 3 h, before they were treated with TVE for 2 h and stimulated for 30 min with nigericin (10 μM) (C), and ATP (5 mM) (D). The level of IL‐1β was evaluated by ELISA and the lysate was analysed by immunoblotting. One representative result of three independent experiments is shown. Values are shown reported as the means of technical triplicates ± SEM. One‐way ANOVA, Bonferroni post‐hoc test; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, n.s. not significant.

Article Snippet: The purified mouse NLRP3 (BPS bioscience, 100189) (0.139 mg/mL) was incubated with TVE (50 μg/mL) or DMSO at 37°C for 30 min in the reaction buffer (100 mM Tris pH 7.8, 2.8 mM EDTA, 100 mM MgCl₂, 15 mM KCl, 655 mM NaCl).

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Nature immunology

Article Title: Anaphylactic degranulation by mast cells requires the mobilization of inflammasome components.

doi: 10.1038/s41590-024-01788-y

Figure Lengend Snippet: Fig. 1 | Inflammasome components mediate MC degranulation following IgE–Ag stimulation. a, Body temperature changes in wild-type (WT), Asc−/− and Nlrp3−/− mice and in KitW-sh/W-sh mice repleted with BMMCs from WT, Asc−/− or Nlrp3−/− mice sensitized with TNP-specific IgE antibody and injected i.v. with TNP-OVA and monitored for 105 min. b, β-hexosaminidase release from DNP- specific IgE-sensitized WT, Asc−/−, Nlrp3−/− and Casp-1/Casp-11−/− BMMCs after exposure to increasing doses of BSA-DNP. c, β-hexosaminidase release from IgE- sensitized and BSA-DNP stimulated RBL-2H3 MCs in which the expression of Asc, Nlrp3 or both was silenced with siRNA and immunoblot analysis of NLRP3 and ASC expression in each condition, with GAPDH as a loading control. d, β-hexosaminidase release from IgE-sensitized, BSA-DNP-stimulated Asc−/− or Nlrp3−/− BMMCs transduced with plasmids encoding mouse ASC, NLRP3 or empty vector (EV) and an immunoblot of endogenous NLRP3 and ASC and Flag–NLRP3 and Flag–ASC in cell lysates of each experimental condition. e, Measurement of intracellular Ca2+ concentrations using Fluo-4 NW assay kit in WT, Asc−/− and Nlrp3−/− BMMCs after IgE–Ag or thapsigargin exposure. f, β-hexosaminidase release from WT, Asc−/− and Nlrp3−/− BMMCs following exposure to thapsigargin

Article Snippet: Ectopic expression of NLRP3 and ASC in Nlrp3−/− or Asc−/− BMMCs BMMCs obtained from either Nlrp3−/− or Asc−/− (2 × 107) or WT were nucleofected with 5 μg of each expression plasmid expressing mouse Flag-tagged NLRP3 (Addgene, 75127) or ASC (Addgene, 75134) or empty plasmid using P3 Primary Cell 4D-Nucleofector X-kit (Lonza).

Techniques: Injection, Expressing, Western Blot, Control, Transduction, Plasmid Preparation

Journal: Nature immunology

Article Title: Anaphylactic degranulation by mast cells requires the mobilization of inflammasome components.

doi: 10.1038/s41590-024-01788-y

Figure Lengend Snippet: Fig. 2 | NLRP3 and ASC form a distinct complex with CD63 on MC granules. a, Immunofluorescence staining of WT BMMCs with CD63 (green), NLRP3 (red, left) or CD63 (green) and ASC (orange, right) at 0, 5 and 10 min following IgE–Ag stimulation. Scale bars, 20 μm. b, Immunoblotting of immunoprecipitated endogenous CD63 with endogenous NLRP3, ASC and caspase-1 in the immunoprecipitation (IP) fractions from untreated or IgE–Ag stimulated WT BMMCs. IgG antibody from the corresponding rabbit or mouse species was used as a control. Protein of interests in the total cell lysates (TCL) are depicted in each fraction. c, Immunoblot analyses of NLRP3, ASC, caspase-1 and CD63 in cell fractions from untreated, IgE–Ag-stimulated or LPS/nigericin-stimulated

Article Snippet: Ectopic expression of NLRP3 and ASC in Nlrp3−/− or Asc−/− BMMCs BMMCs obtained from either Nlrp3−/− or Asc−/− (2 × 107) or WT were nucleofected with 5 μg of each expression plasmid expressing mouse Flag-tagged NLRP3 (Addgene, 75127) or ASC (Addgene, 75134) or empty plasmid using P3 Primary Cell 4D-Nucleofector X-kit (Lonza).

Techniques: Immunofluorescence, Staining, Western Blot, Immunoprecipitation, Control

Journal: Nature immunology

Article Title: Anaphylactic degranulation by mast cells requires the mobilization of inflammasome components.

doi: 10.1038/s41590-024-01788-y

Figure Lengend Snippet: Fig. 3 | NEK7 and Pyk2 kinases are critical initiators of granulosome formation. a, Immunoblotting of NEK7 with NLRP3 from WT BMMCs stimulated with IgE–Ag or left untreated. Protein of interests in the TCL are depicted in each fraction. b, Immunoblot of NEK7 with NLRP3, ASC and CD63 in non-stimulated or IgE–Ag-stimulated WT BMMCs pretreated or not with CAY-10736 or oridonin. Protein of interests in the TCL are depicted in each fraction. c, Immunoblot analysis of cross-linked NLRP3 from IgE–Ag-stimulated or non-stimulated WT BMMCs pretreated or not with CAY-10736 or oridonin. d, Immunoblot analysis of CD63 interaction with NEK7, NLRP3 and ASC in unstimulated or IgE–Ag-stimulated WT BMMCs pretreated or not with CAY-10736 or oridonin. The proteins of interest in the TCL are depicted in each fraction. e, Secretion of β-hexosaminidase in WT and Nlrp3−/− BMMCs pretreated with vehicle, CAY- 10736 or oridonin and stimulated with IgE–Ag. f, Immunoblot analysis of PyK2 interaction with ASC in WT BMMCs at 0, 3, 5, 15 and 30 min after stimulation with IgE–Ag. The proteins of interest in the TCL are depicted in each fraction. g, Immunoblot analysis of PyK2 interactions with ASC, CD63 and NLRP3 in

Article Snippet: Ectopic expression of NLRP3 and ASC in Nlrp3−/− or Asc−/− BMMCs BMMCs obtained from either Nlrp3−/− or Asc−/− (2 × 107) or WT were nucleofected with 5 μg of each expression plasmid expressing mouse Flag-tagged NLRP3 (Addgene, 75127) or ASC (Addgene, 75134) or empty plasmid using P3 Primary Cell 4D-Nucleofector X-kit (Lonza).

Techniques: Western Blot

Journal: Nature immunology

Article Title: Anaphylactic degranulation by mast cells requires the mobilization of inflammasome components.

doi: 10.1038/s41590-024-01788-y

Figure Lengend Snippet: Fig. 4 | Granulosomes facilitate MT polymerization and granule trafficking. a, Immunofluorescence microscopy of α-tubulin staining in WT, Nlrp3−/− and Asc−/− BMMCs before and 10 min after IgE–Ag stimulation. Scale bars, 20 μm. b, Protein immunoblot analysis of insoluble and Triton buffer soluble fractions in WT, Nlrp3−/−, Asc−/− and Casp-1/11−/− BMMCs stimulated or not with IgE–Ag with SDS–PAGE gel stained with Coomassie brilliant blue used as an equal loading control. c, Immunoblot analysis of NLRP3, ASC or CD63 interactions with dynein in WT BMMCs stimulated with IgE–Ag for 0, 5 or 10 min. The proteins in the TCL are depicted in each fraction. d, Immunoblot analysis of CD63 interactions with

Article Snippet: Ectopic expression of NLRP3 and ASC in Nlrp3−/− or Asc−/− BMMCs BMMCs obtained from either Nlrp3−/− or Asc−/− (2 × 107) or WT were nucleofected with 5 μg of each expression plasmid expressing mouse Flag-tagged NLRP3 (Addgene, 75127) or ASC (Addgene, 75134) or empty plasmid using P3 Primary Cell 4D-Nucleofector X-kit (Lonza).

Techniques: Immunofluorescence, Microscopy, Staining, Western Blot, SDS Page, Control

Journal: Nature immunology

Article Title: Anaphylactic degranulation by mast cells requires the mobilization of inflammasome components.

doi: 10.1038/s41590-024-01788-y

Figure Lengend Snippet: Fig. 5 | NLRP3-targeting drug protects against anaphylaxis. a, β-hexosaminidase release in WT, Nlrp3−/− and Asc−/− BMMCs pretreated with 0, 2,10 and 50 μM of CY-09 and stimulated with IgE–Ag (TNP-OVA). b, Body temperature changes at 0, 30, 60, 90 and 120 min after Ag administration in IgE- sensitized KitW-sh/W-sh mice repleted with WT, Asc−/− or Nlrp3−/− BMMCs pretreated or not with CY-09 for 1 h. c, Immunoblot analysis of CD63 interactions with NLRP3, ASC and tubulin in non-stimulated or IgE–Ag-stimulated WT BMMCs pretreated or not with CY-09. The proteins of interest in TCL are depicted in each fraction. d, β-hexosaminidase release from Nlrp3 KD or Asc KD human LAD2 MCs pretreated or not with CY-09 and stimulated or not with IgE–Ag and immunoblots showing NLRP3 and ASC protein expression in TCL. Data

Article Snippet: Ectopic expression of NLRP3 and ASC in Nlrp3−/− or Asc−/− BMMCs BMMCs obtained from either Nlrp3−/− or Asc−/− (2 × 107) or WT were nucleofected with 5 μg of each expression plasmid expressing mouse Flag-tagged NLRP3 (Addgene, 75127) or ASC (Addgene, 75134) or empty plasmid using P3 Primary Cell 4D-Nucleofector X-kit (Lonza).

Techniques: Western Blot, Expressing

Journal: Nature immunology

Article Title: Anaphylactic degranulation by mast cells requires the mobilization of inflammasome components.

doi: 10.1038/s41590-024-01788-y

Figure Lengend Snippet: Fig. 6 | Pro-IL-1β is released from intact granules following IgE–Ag activation of LPS-primed MCs. a, ELISA showing release of IL-1β or β-hexosaminidase from WT or Nlrp3−/− BMMCs pretreated or not with TNP-specific IgE and LPS followed by stimulation with OVA-TNP (Ag) or not. b, Immunofluorescence staining of IL-1β (red) and CD63 (green) in WT or Nlrp3−/− BMMCs primed with LPS and TNP-specific IgE for 3–4 h and stimulated with OVA-TNP (Ag) at 0, 5 and 10 min (WT) or 10 min (Nlrp3−/−) and colocalization of IL-1β with CD63 calculated from 13 representative images using ImageJ (Pearson correlation coefficient). Values are the mean ± s.e.m.; significant differences of treatment versus control were analyzed by one-way ANOVA/Bonferroni’s post hoc test, ***P < 0.001, n = 13 images per group. Scale bars, 20 μm. c, Immunoblot analysis of pro-IL-1β and mature IL-1β in concentrated supernatants of WT or Nlrp3−/− BMMCs activated with IgE–Ag alone or LPS + IgE–Ag and Nlrp3−/− BMMCs transfected with Flag or Flag-tagged mouse NLRP3. d, Immunoblot analysis of pro-IL-1β and mature IL-1β in the supernatant of non-primed or LPS + IgE-primed Ag-activated WT BMMCs pretreated or not with chymostatin. e, Immunoblot analysis of pro-IL-1β and mature IL-1β in the supernatant and granule remnant fractions of WT BMMCs primed or not with LPS and stimulated or not with Ag. f, Body temperature changes at 0, 30, 60, 90 and 120 min after Ag administration in WT mice pre- sensitized with TNP-specific IgE antibody (5 µg per mouse) for 2–4 days before

Article Snippet: Ectopic expression of NLRP3 and ASC in Nlrp3−/− or Asc−/− BMMCs BMMCs obtained from either Nlrp3−/− or Asc−/− (2 × 107) or WT were nucleofected with 5 μg of each expression plasmid expressing mouse Flag-tagged NLRP3 (Addgene, 75127) or ASC (Addgene, 75134) or empty plasmid using P3 Primary Cell 4D-Nucleofector X-kit (Lonza).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Control, Western Blot, Transfection