Journal: EMBO Molecular Medicine

Article Title: Tranilast directly targets NLRP 3 to treat inflammasome‐driven diseases

doi: 10.15252/emmm.201708689

Figure Lengend Snippet: Immunoblot analysis of ASC oligomerization in lysates of BMDMs treated with various doses (above lanes) of TR for 30 min and then stimulated with nigericin for another 30 min. Immunoprecipitation (IP) and immunoblot analysis of the interaction of endogenous NLRP3 and NEK7 in LPS‐primed BMDMs treated with various doses (above lanes) of TR for 30 min and then stimulated with nigericin for another 20 min. IP and immunoblot analysis of the interaction of endogenous NLRP3 and ASC in LPS‐primed BMDMs treated with various doses (above lanes) of TR for 30 min and then stimulated with nigericin for another 20 min. Data information: Data are representative of two or three independent experiments. Source data are available online for this figure.

Article Snippet: Recombinant human NLRP3 was from Novus Biologicals.

Techniques: Western Blot, Immunoprecipitation

Journal: EMBO Molecular Medicine

Article Title: Tranilast directly targets NLRP 3 to treat inflammasome‐driven diseases

doi: 10.15252/emmm.201708689

Figure Lengend Snippet: A, B Cell lysates of LPS‐primed BMDMs (A) or PMA‐differentiated THP‐1. (B) Cells were incubated with different concentrations of biotin‐TR for 1 h, which were then pulled down with streptavidin beads. C Purified recombinant NLRP3 protein was incubated with different concentrations of biotin‐TR for 1 h, which were then pulled down with streptavidin beads. D, E Cell lysates from HEK‐293T cells transfected with Flag‐tagged NLRP3, NOD1, NOD2, NLRP1, NLRC4, NLRP3‐LRR, NLPR3‐NACHT, or NLRP3‐PYD constructs were incubated with different concentrations of biotin‐TR for 1 h, which were then pulled down with streptavidin beads. F Immunoprecipitation (IP) and immunoblot analysis of the interaction of Flag‐NLRP3 and VSV‐NLRP3 in the lysates of HEK‐293T cells. TR was added at 8 h post‐transfection. G Immunoblot analysis of NLRP3 by SDD‐AGE or SDS–PAGE assay in BMDMs treated with TR for 30 min and then left stimulated with nigericin for 20 min. Data information: Data are representative of two or three independent experiments. Source data are available online for this figure.

Article Snippet: Recombinant human NLRP3 was from Novus Biologicals.

Techniques: Incubation, Purification, Recombinant, Transfection, Construct, Immunoprecipitation, Western Blot, SDS Page

Journal: EMBO Molecular Medicine

Article Title: Tranilast directly targets NLRP 3 to treat inflammasome‐driven diseases

doi: 10.15252/emmm.201708689

Figure Lengend Snippet: A, B FACS analysis of neutrophil numbers (A) or ELISA (B) of IL‐1β in the peritoneal cavity of 10‐week‐old male C57BL/6J mice intraperitoneally injected with MSU (1 mg/mouse) with or without TR (200 mg/kg of body weight). n = 8 per group. C, D Time course of changes of joint swelling (C) or ELISA (D) of IL‐1β in the supernatants of joint culture of 10‐week‐old male WT or Nlrp3 −/− mice intra‐articularly injected with MSU with or without oral TR administration (200 mg/kg of body weight). n = 6 per group. E Nlrp3A 350VneoR crossed with LysM‐Cre mice (NLRP3‐mut) treated with vehicle or TR at day 9. F Weight of wild‐type mice or Nlrp3A 350VneoR crossed with LysM‐Cre mice (NLRP3‐mut) treated with vehicle or TR at day 9. For WT vehicle and WT TR, n = 6; NLRP3‐mut PBS n = 4; and NLRP3‐mut TR n = 8. G Survival of NLRP3‐mut mice treated with vehicle or TR up to day 18. TR group n = 8, vehicle group n = 7. Data information: Data are shown as mean and s.e.m. and are representative of two or three independent experiments. Statistics were analyzed using an unpaired Student's t ‐test or a generalized Wilcoxon test (G): * P < 0.05, *** P < 0.001, NS, not significant.

Article Snippet: Recombinant human NLRP3 was from Novus Biologicals.

Techniques: Enzyme-linked Immunosorbent Assay, Injection

Journal: EMBO Molecular Medicine

Article Title: Tranilast directly targets NLRP 3 to treat inflammasome‐driven diseases

doi: 10.15252/emmm.201708689

Figure Lengend Snippet: A Body weights change of 6‐week‐old male wild‐type (WT) or Nlrp3 −/− mice that were first fed with HFD for 14 weeks and then treated with different dose of oral TR for 6 weeks. n = 6 per group. B, C Fed (B) or fasting (C) blood glucose concentrations at week 6 in the mice described in (A). n = 6. D–G ITT (D, F) or GTT (E, G) performed at week 6 in the mice described in (A). n = 6 per group. Data information: Data are shown as mean and s.e.m. and are representative of two independent experiments. Statistics were analyzed using an unpaired Student's t ‐test or two‐way ANOVA for (D–G): * P < 0.05, ** P < 0.01, *** P < 0.001, NS, not significant.

Article Snippet: Recombinant human NLRP3 was from Novus Biologicals.

Techniques:

Journal: Frontiers in Immunology

Article Title: Cell type-specific roles of NLRP3, inflammasome-dependent and -independent, in host defense, sterile necroinflammation, tissue repair, and fibrosis

doi: 10.3389/fimmu.2023.1214289

Figure Lengend Snippet: Some experimental studies reporting expression and function of the NLRP3 inflammasome in non-myeloid cells.

Article Snippet: The Human Protein Atlas reports transcript expression under basal conditions in endothelial cells from different organs that do not appear to express relevant levels of NLRP3 mRNA , although they do express ASC and CASP1 .

Techniques: Expressing, Cell Differentiation

Journal: Frontiers in Immunology

Article Title: Cell type-specific roles of NLRP3, inflammasome-dependent and -independent, in host defense, sterile necroinflammation, tissue repair, and fibrosis

doi: 10.3389/fimmu.2023.1214289

Figure Lengend Snippet: Inflammasome-dependent and –independent roles of NLRP3 in myeloid cells. Left: Canonical activation of NLRP3 starts with TLR activation, mediate by Myd88 and NF-κB, which triggers the expression of NLRP3, pro-IL-1β and -IL-18. Several stimuli can trigger the inflammasome assembly, such as K + efflux, Ca +2 influx, crystals, phagosome rupture, mtROS and mtDNA. The conjunction of NLRP3, ASC and Caspase 1 lead to Caspase 1 activation and proteolytic cleavage of pro-IL-1β and -IL-18, as well GSDMD resulting in IL-1β and -IL-18 secretion and/or pyroptosis. Right: Non-canonical NLRP3 activation is triggered by TLR activation, via Myd88 and TRIF, or type I interferon receptor (IFRA), which lead to NF-κB activation and induction of NLRP3, pro-IL-1β and -IL-18 expression. The type I interferon triggers caspase-11, and caspase 1 expression. LPS and LPG can bind and directly activate caspase 11, which cleaves GSDMD in GSDMD-N driving pyroptosis. Nlrp3 can act as a transcription factor in T cells by binding directly to the Il4 , Il13 , Icos , Maf , Il1r1 promoter region. Nlrp3 can induce phosphorylation of Smad2/4 in the TGFβR pathway, thereby regulating the transcription of target genes. Created with BioRender.com .

Article Snippet: The Human Protein Atlas reports transcript expression under basal conditions in endothelial cells from different organs that do not appear to express relevant levels of NLRP3 mRNA , although they do express ASC and CASP1 .

Techniques: Activation Assay, Expressing, Binding Assay, Phospho-proteomics

Journal: Frontiers in Immunology

Article Title: Cell type-specific roles of NLRP3, inflammasome-dependent and -independent, in host defense, sterile necroinflammation, tissue repair, and fibrosis

doi: 10.3389/fimmu.2023.1214289

Figure Lengend Snippet: Reported inflammasome-dependent and -independent functions of NLRP3 in non-immune cells. In T cells, canonical activation of NLRP3 has been described to favor differentiation of naive T cells into Th17 cells, as well as to increase IFN-γ production in the face of viral infection. In an inflammasome independent way, the NLRP3 acts as a transcription factor in T cells, favoring a Th2 profile. In epithelial cells, canonical activation of NLRP3 is related to the production and release of IL-1β and IL-18 during inflammatory processes, whereas the non-canonical pathway favors the maintenance of the intestinal epithelial barrier. In epithelial cells the inflammasome independent function is related with TGF-β pathway activation and fibrosis, as well acting as a transcription factor controlling the expression of IL-33. The role of NLRP3 in endothelial cells has been pointed out during inflammatory processes, in which IL-1 β production favors endothelial dysfunction and increased expression of selectins, acting on the recruitment of inflammatory cells to the injured site. In B cells NLRP3 stimulates the IgM production, as well the expression of CXCR4 and CCR7, homing related chemokines. *EpC (epithelial cell). *EnC (endothelial cell). Created with BioRender.com .

Article Snippet: The Human Protein Atlas reports transcript expression under basal conditions in endothelial cells from different organs that do not appear to express relevant levels of NLRP3 mRNA , although they do express ASC and CASP1 .

Techniques: Activation Assay, Infection, Expressing