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

Article Title: DDX3X acts as a live-or-die checkpoint in stressed cells by regulating NLRP3 inflammasome

doi: 10.1038/s41586-019-1551-2

Figure Lengend Snippet: a, Co-immunoprecipitation (IP) of NLRP3 and DDX3X in BMDMs treated with LPS with or without nigericin. Representative blots (n= 3). b, Immunoblot analysis of CASP1 cleavage in wild-type (WT), Ddx3xfl/fl, Ddx3xfl/flLysMcre and Nlrp3−/− BMDMs treated with LPS and nigericin. Representative blots (n > 3). c, Immunoblots of DDX3X expression and CASP1 cleavage after siRNA-mediated knockdown of Ddx3x in BMDMs treated with LPS with or without nigericin. Representative blots (n = 2). d, Confocal microscopy imaging of ASC specks in BMDMs treated with LPS with or without nigericin, to visualize the subcellular localization of DDX3X, NLRP3 and ASC. Scale bars, 10 μm. Representative images (n = 3). e, Schematic of N-terminal Flag-tagged NLRP3 expression constructs: full-length NLRP3 (Flag-NLRP3-FL), the pyrin domain of NLRP3 (Flag-PYD; amino acids 1–90 of NLRP3), the NACHT domain of NLRP3 (Flag-NACHT; amino acids 91–710 of NLRP3) and the LRR domain of NLRP3 (Flag-LRR; amino acids 711–1034 of NLRP3). f, Immunoblot (IB) analysis of input lysates used for co-immunoprecipitation of Flag-tagged NLRP3 constructs and DDX3X-mCherry. Representative blots (n > 3). g, Immunoblot analysis of immunoprecipitation of DDX3X-mCherry and the indicated NLRP3 constructs. Red asterisk indicates the antibody light chain. Representative blots (n > 3).

Article Snippet: NLRP3 expression constructs pCDNA3-N-Flag-NLRP3, pCDNA3-N-Flag-NLRP3 1–90, pCDNA3-N-Flag-NLRP3 91–710 and pCDNA3-N-Flag-NLRP3 711–1034 were purchased from AddGene.

Techniques: Immunoprecipitation, Western Blot, Expressing, Confocal Microscopy, Imaging, Construct

Journal: Nature

Article Title: DDX3X acts as a live-or-die checkpoint in stressed cells by regulating NLRP3 inflammasome

doi: 10.1038/s41586-019-1551-2

Figure Lengend Snippet: a, Immunoblot analysis of lysates fractionated by analytical ultra-centrifugation to test the oligomerization of NLRP3, DDX3X and ASC in BMDMs stimulated with LPS and treated either with nigericin or with arsenite and nigericin. Representative blots (n = 2). MW, molecular weight. b, Immunoblot analysis of ASC from the insoluble fraction of samples from a under non-reducing (without β-mercaptoethanol; – BME) and reducing (+BME) conditions. Representative blots (n = 2). c, STORM imaging of ASC speck assembly and its spatial organization with DDX3X over time. Scale bars, 5 μm (whole-cell images); 1 μm (magnified images). Representative images (n = 2). d, Plot of the r-index to show the extent of colocalization of ASC and DDX3X with the duration of nigericin treatment (n = 2). R2 was calculated using linear regression analysis; P = 0.0125 (for the significance of the slope being non-zero). Data are mean ± s.e.m. e, f, Immunoblot analysis of CASP1 cleavage in LPS-primed BMDMs to which arsenite was added at various time points (30 and 15 min before adding nigericin, simultaneously with nigericin and 15 and 30 min after adding nigericin), without (e) or with (f) pre-treatment with anisomycin. Representative blots (n = 3). g, h, Structured illumination microscopy of BMDMs to visualize the subcellular localization of DDX3X, G3BP1 and ASC in LPS-primed BMDMs treated with nigericin alone (g) or with arsenite and nigericin (h). Scale bars, 3 μm. Representative images (n = 3).

Article Snippet: NLRP3 expression constructs pCDNA3-N-Flag-NLRP3, pCDNA3-N-Flag-NLRP3 1–90, pCDNA3-N-Flag-NLRP3 91–710 and pCDNA3-N-Flag-NLRP3 711–1034 were purchased from AddGene.

Techniques: Western Blot, Centrifugation, Molecular Weight, Imaging, Microscopy

Journal: Nature

Article Title: DDX3X acts as a live-or-die checkpoint in stressed cells by regulating NLRP3 inflammasome

doi: 10.1038/s41586-019-1551-2

Figure Lengend Snippet: a, Confocal microscopy imaging of peritoneal CD45+ cells to visualize in vivo stress granules in Ddx3xfl/fl and Ddx3xfl/flLysMcre mice that were treated with PBS or arsenite. Scale bars, 10 μm. Representative images (n = 2). b, Quantification of CD45+ cells that contain stress granules, from the peritoneal cavity of Ddx3xfl/fl and Ddx3xfl/flLysMcre mice that were injected with arsenite or PBS. ****P < 0.0001 (unpaired two-sided t-test). Data represent two biologically independent experiments (n = 10 frames). c, Quantification of the numbers and percentage of CD11b+ myeloid cells in the peritoneal cavity. P values (from left to right): ***P = 0.0001, ***P = 0.0005 (unpaired two-sided t-test; n = 7). d, e, Levels of IL-1β in the serum (d) and peritoneal fluid (PF) (e) of mice that were injected with LPS with or without prior arsenite injection in the peritoneum. P values: **P = 0.0026 (d), P = 0.09 (e) (unpaired two-sided t-test; n = 7). f, Levels of IL-1β in the serum and peritoneal fluid of Ddx3xfl/fl and Ddx3xfl/flLysMcre mice that were injected with LPS in the peritoneum. P values (from top to bottom): **P = 0.0073, *P = 0.0123 (unpaired two-sided t-test; n ≥ 6). Data are mean ± s.e.m. (b-f). g, Schematic of the interplay between the inflammasome and stress granules that is involved in cell-fate decisions. DDX3X promotes NLRP3 inflammasome activation and the pro-death cell-fate decision probably by interacting with the NLRP3 NACHT domain through its helicase (Heli) domain. Induction of stress granules causes the sequestration of DDX3X (along with 40S ribosomal subunits and translation initiation factors (eIFs)), thus making it unavailable for NLRP3 inflammasome activation and thereby allowing the cells to make a pro-survival cell-fate choice.

Article Snippet: NLRP3 expression constructs pCDNA3-N-Flag-NLRP3, pCDNA3-N-Flag-NLRP3 1–90, pCDNA3-N-Flag-NLRP3 91–710 and pCDNA3-N-Flag-NLRP3 711–1034 were purchased from AddGene.

Techniques: Confocal Microscopy, Imaging, In Vivo, Injection, Activation Assay