Journal: Cell death & disease

Article Title: Clinical-mediated discovery of pyroptosis in CD8 + T cell and NK cell reveals melanoma heterogeneity by single-cell and bulk sequence.

doi: 10.1038/s41419-023-06068-5

Figure Lengend Snippet: Fig. 2 Melanoma specimens reduce PRGs positive cells in epidermal clinically. A Overall survival analysis based on K-M was calculated to identify the potential PRGs clinically. B PPI via MCODE and CytoNCA algorithm was established, in which PRGs were screened out as central protein functioning in melanoma deficiency. C GZMA, GSDMB, CHMP4A, NLRP1, and IL18 protein expression of melanoma and control patients detected by ELISA. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n = 5, compared with the normal group. D Representative IHC stanning of GZMA+, GSMDB+, CHMP4A+, NLRP1+, and IL18+ in melanoma specimens from the clinic. Scale bar = 100 μm, n = 5 samples per group. E The quantification of PRGs protein in epidermal areas, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, compared with the control group.

Article Snippet: PRGs protein expression detected by ELISA Human CHMP4A ELISA-kit (CSB-CL883630HU2, CUSABIO), human GSDMB ELISAkit (PH103310, PYRAM), human GZMA ELISA-kit (ab225728, Abcam), human IL18 ELISA-kit (ab215539, Abcam), and human NLRP1 ELISA-kit (CSB-EL015864HU, CUSABIO) were used to monitor the kernel PRGs’ protein content.

Techniques: Expressing, Control, Enzyme-linked Immunosorbent Assay

Journal: Cell death & disease

Article Title: Clinical-mediated discovery of pyroptosis in CD8 + T cell and NK cell reveals melanoma heterogeneity by single-cell and bulk sequence.

doi: 10.1038/s41419-023-06068-5

Figure Lengend Snippet: Fig. 5 PRGs were primarily expressed in immune cells. A Feature plots depicting the expression of key pyroptosis, violin plots were also displayed to determine the cell type. B Representative IHC stanning of GZMA+, GSMDB+, CHMP4A+, NLRP1+, and IL18+ in melanoma specimens, especially in lymphocyte areas. Scale bar = 100 μm, n = 4 samples per group. C The quantification of PRGs protein in dermal areas, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, compared with the control group.

Article Snippet: PRGs protein expression detected by ELISA Human CHMP4A ELISA-kit (CSB-CL883630HU2, CUSABIO), human GSDMB ELISAkit (PH103310, PYRAM), human GZMA ELISA-kit (ab225728, Abcam), human IL18 ELISA-kit (ab215539, Abcam), and human NLRP1 ELISA-kit (CSB-EL015864HU, CUSABIO) were used to monitor the kernel PRGs’ protein content.

Techniques: Expressing, Control

Journal: bioRxiv

Article Title: DPP9 directly sequesters the NLRP1 C-terminus to repress inflammasome activation

doi: 10.1101/2020.08.14.246132

Figure Lengend Snippet: a , Domain organization. b , SDS-PAGE of the purified NLRP1-DPP9 complex. HSP70 contamination is noted with an asterisk (*). c , Cryo-EM map (left) and the model (right) of the ternary NLRP1 A -NLRP1 B -DPP9 complex. The DPP9 dimer and the two copies of NLRP1 (A and B) are labelled with the colour scheme in (a). A schematic diagram (middle) denotes the entire NLRP1 and DPP9 molecules versus the ordered, resolved portions of the proteins (red circle). d , FIIND A overview with ZU5 (blue) and UPA (light pink) subdomains. The catalytic triad residues (H1186, E1195, and S1213) are shown in sticks. e , Topology of the FIIND with secondary structures labelled. f , Superimposition of FIIND A onto the UPA B . NLRP1 B has to be free NLRP1-CT because a ZU5 subdomain at site B would have clashed with ZU5 and UPA at site A and with DPP9.

Article Snippet: Primary antibodies used in this study include: DPP9 rabbit polyclonal Ab (Abcam, Ab42080), FLAG® M2 mouse monoclonal Ab (Sigma, F3165), GAPDH rabbit monoclonal Ab (Cell Signaling Technology, 14C10), NLRP1 mouse monoclonal Ab (R&D Systems, MAB6788), CASP1 rabbit polyclonal Ab (Cell Signaling Technology, 2225), GSDMDC1 rabbit polyclonal Ab (Novus, NBP2-33422), V5 rabbit polyclonal Ab (Abcam, Ab9116).

Techniques: SDS Page, Purification, Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: DPP9 directly sequesters the NLRP1 C-terminus to repress inflammasome activation

doi: 10.1101/2020.08.14.246132

Figure Lengend Snippet: a , Purification of the NLRP1-DPP9 complex by ion exchange chromatography. The ternary complex peak is shaded in green and labelled with an arrow. b , A representative cryo-EM micrograph. c , Representative 2D class averages. d , Workflow for the NLRP1-DPP9 complex structure determination. e , Map-map and map-model FSC curves. f , Local resolution distribution of the final map calculated with ResMap .

Article Snippet: Primary antibodies used in this study include: DPP9 rabbit polyclonal Ab (Abcam, Ab42080), FLAG® M2 mouse monoclonal Ab (Sigma, F3165), GAPDH rabbit monoclonal Ab (Cell Signaling Technology, 14C10), NLRP1 mouse monoclonal Ab (R&D Systems, MAB6788), CASP1 rabbit polyclonal Ab (Cell Signaling Technology, 2225), GSDMDC1 rabbit polyclonal Ab (Novus, NBP2-33422), V5 rabbit polyclonal Ab (Abcam, Ab9116).

Techniques: Purification, Ion Exchange Chromatography, Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: DPP9 directly sequesters the NLRP1 C-terminus to repress inflammasome activation

doi: 10.1101/2020.08.14.246132

Figure Lengend Snippet: a , Summary of BS3 crosslinking between DPP9 and NLRP1. High confidence crosslinked peptides are displayed, with residue ranges labelled and colours coded by domains. Crosslinked lysine pairs are indicated in red. Cα-Cα distances between these lysine residues interpreted by the final NLRP1-DPP9 model are shown, along with the figure panel names for their detailed depictions. b , Overview of BS3-mediated crosslinks. c-h , Zoom-ins highlighting crosslinked lysine pairs (red) interpreted by the final NLRP1-DPP9 model.

Article Snippet: Primary antibodies used in this study include: DPP9 rabbit polyclonal Ab (Abcam, Ab42080), FLAG® M2 mouse monoclonal Ab (Sigma, F3165), GAPDH rabbit monoclonal Ab (Cell Signaling Technology, 14C10), NLRP1 mouse monoclonal Ab (R&D Systems, MAB6788), CASP1 rabbit polyclonal Ab (Cell Signaling Technology, 2225), GSDMDC1 rabbit polyclonal Ab (Novus, NBP2-33422), V5 rabbit polyclonal Ab (Abcam, Ab9116).

Techniques: Residue

Journal: bioRxiv

Article Title: DPP9 directly sequesters the NLRP1 C-terminus to repress inflammasome activation

doi: 10.1101/2020.08.14.246132

Figure Lengend Snippet: a , ClustalW multiple sequence alignment between human NLRP1 (hNLRP1), mouse NLRP1 (mNLRP1, different isoforms), and rat NLRP1 (rNLRP1, different isoforms). COP: Copenhagen; ZUC: Zucker; LEW: Lewis; SD: Sprague Dawley; and CDF: Fischer. Secondary structures and residue numbers are denoted based on the human FIIND A structure in the NLRP1-DPP9 ternary complex. Interfacial residues in the NLRP1-DPP9 complex are annotated with asterisks, and residues in the catalytic triad (H1186, E1195, S1213) are boxed in black. b , The ZU5 A -UPA A -UPA B module that binds DPP9. UPA A and UPA B interact with each other in a front-to-back manner with only a 9° rotation between them. c , Altered conformation of the UPA B N-terminus that binds in the DPP9 active site tunnel in comparison to UPA A in a complete FIIND A .

Article Snippet: Primary antibodies used in this study include: DPP9 rabbit polyclonal Ab (Abcam, Ab42080), FLAG® M2 mouse monoclonal Ab (Sigma, F3165), GAPDH rabbit monoclonal Ab (Cell Signaling Technology, 14C10), NLRP1 mouse monoclonal Ab (R&D Systems, MAB6788), CASP1 rabbit polyclonal Ab (Cell Signaling Technology, 2225), GSDMDC1 rabbit polyclonal Ab (Novus, NBP2-33422), V5 rabbit polyclonal Ab (Abcam, Ab9116).

Techniques: Sequencing, Residue, Comparison

Journal: bioRxiv

Article Title: DPP9 directly sequesters the NLRP1 C-terminus to repress inflammasome activation

doi: 10.1101/2020.08.14.246132

Figure Lengend Snippet: a , Insertion of the N-terminal peptide of UPA B into the DPP9 active site. b , Displacement of the UPA B N-terminal peptide from the DPP9 active site by VbP. c , Cryo-EM map of the NLRP1-DPP9 complex in the presence of VbP. VbP binding reduces UPA B occupancy. d , Ability of DPP9 to cleave an isolated UPA N-terminal peptide, but not an entire UPA. e , Comparison of the binding modes of the UPA B N-terminal peptide in the NLRP1-DPP9 complex and the Ile-Pro dipeptide in an acyl-enzyme intermediate . f , Overview of three interfaces important for NLRP1-DPP9 association. Regions blocked in rectangles are shown in detail in (a) and (g-i). g-i , Zoom-ins of each NLRP1-DPP9 binding interface. j , FLAG co-immunoprecipitation using FLAG-tagged WT NLRP1 and His-tagged WT and mutant DPP9. Anti-DPP9, anti-NLRP1-CT (NLRP1-FL and NLRP1-CT), and anti-GAPDH antibodies were used in the immunoblots. EV: empty vector. The Roman numerals in the parentheses represent the three interfaces in the NLRP1-DPP9 ternary complex. Each immunoblot is representative of > 2 independent experiments. k , FLAG co-immunoprecipitation using FLAG-tagged WT or mutant NLRP1 and His-tagged WT DPP9. FLAG-tagged GFP was used as a negative control. Anti-DPP9, anti-FLAG (NLRP1-FL, NLRP1-CT, and GFP), and anti-GAPDH antibodies were used in the immunoblots. The Roman numerals in parentheses represent the three interfaces in the NLRP1-DPP9 ternary complex. Each immunoblot is representative of > 2 independent experiments.

Article Snippet: Primary antibodies used in this study include: DPP9 rabbit polyclonal Ab (Abcam, Ab42080), FLAG® M2 mouse monoclonal Ab (Sigma, F3165), GAPDH rabbit monoclonal Ab (Cell Signaling Technology, 14C10), NLRP1 mouse monoclonal Ab (R&D Systems, MAB6788), CASP1 rabbit polyclonal Ab (Cell Signaling Technology, 2225), GSDMDC1 rabbit polyclonal Ab (Novus, NBP2-33422), V5 rabbit polyclonal Ab (Abcam, Ab9116).

Techniques: Cryo-EM Sample Prep, Binding Assay, Isolation, Comparison, Immunoprecipitation, Mutagenesis, Western Blot, Plasmid Preparation, Negative Control

Journal: bioRxiv

Article Title: DPP9 directly sequesters the NLRP1 C-terminus to repress inflammasome activation

doi: 10.1101/2020.08.14.246132

Figure Lengend Snippet: a , Purification of the NLRP1-DPP9 complex in the presence of VbP by ion exchange chromatography. The ternary complex peak is shaded in green and labelled with an arrow. b , A representative cryo-EM micrograph. c , Representative 2D class averages. d , Workflow for the NLRP1-DPP9-VbP complex structure determination. e , Map-map and map-model FSC curves. f , Local resolution distribution of the final map calculated with ResMap .

Article Snippet: Primary antibodies used in this study include: DPP9 rabbit polyclonal Ab (Abcam, Ab42080), FLAG® M2 mouse monoclonal Ab (Sigma, F3165), GAPDH rabbit monoclonal Ab (Cell Signaling Technology, 14C10), NLRP1 mouse monoclonal Ab (R&D Systems, MAB6788), CASP1 rabbit polyclonal Ab (Cell Signaling Technology, 2225), GSDMDC1 rabbit polyclonal Ab (Novus, NBP2-33422), V5 rabbit polyclonal Ab (Abcam, Ab9116).

Techniques: Purification, Ion Exchange Chromatography, Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: DPP9 directly sequesters the NLRP1 C-terminus to repress inflammasome activation

doi: 10.1101/2020.08.14.246132

Figure Lengend Snippet: a , N-terminal sequencing of the purified NLRP1-DPP9 complex showing that the NLRP1-CT is not cleaved by the co-expressed DPP9. b , CHOPS assay showing that DPP9 does not cleave NLRP1-CT. Briefly, WT NLRP1 expressed in DPP8/9 DKO HEK293T cells was incubated with PBS or recombinant DPP9 prior to labelling with the 2PCA biotin probe to capture biotinylated proteins. The inputs and the eluents were analysed by immunoblots using anti-NLRP1-CT (NLRP1-FL and NLRP1-CT), anti-GAPDH, and anti-Biotin antibodies. c , Evidence of cleavage of the isolated 15 residue N-terminal peptide in NLRP1-CT by recombinant DPP9 from mass spectrometry analysis. d , Inhibition of DPP9 catalytic activity against AP-AMC by the isolated NLRP1-CT peptide. All data are representative of > 2 independent experiments.

Article Snippet: Primary antibodies used in this study include: DPP9 rabbit polyclonal Ab (Abcam, Ab42080), FLAG® M2 mouse monoclonal Ab (Sigma, F3165), GAPDH rabbit monoclonal Ab (Cell Signaling Technology, 14C10), NLRP1 mouse monoclonal Ab (R&D Systems, MAB6788), CASP1 rabbit polyclonal Ab (Cell Signaling Technology, 2225), GSDMDC1 rabbit polyclonal Ab (Novus, NBP2-33422), V5 rabbit polyclonal Ab (Abcam, Ab9116).

Techniques: Sequencing, Purification, Incubation, Recombinant, Western Blot, Isolation, Residue, Mass Spectrometry, Inhibition, Activity Assay

Journal: bioRxiv

Article Title: DPP9 directly sequesters the NLRP1 C-terminus to repress inflammasome activation

doi: 10.1101/2020.08.14.246132

Figure Lengend Snippet: a , Disorder to order transition of several DPP9 surface loops from the isolated DPP9 crystal structure (PDB ID: 6EOQ) to the NLRP1-bound DPP9 cryo-EM structure. b , Genomic confirmation of DPP8 KO in DPP8/9 DKO HEK293T cells. Single guide RNA (sgRNA) sequence is highlighted. c , Immunoblots of the input lysates for the FLAG co-immunoprecipitation with WT or mutant DPP9 and WT NLRP1-FLAG. d , Cleavage rate of a model DPP9 substrate, GP-AMC, by WT DPP9 and its structure-guided mutants. Only the catalytically dead mutant S730A disrupts catalytic activity and sensitivity to the DPP9 inhibitor, VbP. e , Immunoblots of the input lysates for the FLAG co-immunoprecipitation with WT or mutant NLRP1-FLAG and WT DPP9. All data are representative of > 2 independent experiments.

Article Snippet: Primary antibodies used in this study include: DPP9 rabbit polyclonal Ab (Abcam, Ab42080), FLAG® M2 mouse monoclonal Ab (Sigma, F3165), GAPDH rabbit monoclonal Ab (Cell Signaling Technology, 14C10), NLRP1 mouse monoclonal Ab (R&D Systems, MAB6788), CASP1 rabbit polyclonal Ab (Cell Signaling Technology, 2225), GSDMDC1 rabbit polyclonal Ab (Novus, NBP2-33422), V5 rabbit polyclonal Ab (Abcam, Ab9116).

Techniques: Isolation, Cryo-EM Sample Prep, Sequencing, Western Blot, Immunoprecipitation, Mutagenesis, Activity Assay

Journal: bioRxiv

Article Title: DPP9 directly sequesters the NLRP1 C-terminus to repress inflammasome activation

doi: 10.1101/2020.08.14.246132

Figure Lengend Snippet: Interface I and II mutants cause NLRP1 autoactivation and UPA-UPA interactions are required for inflammasome activity. a , LDH release (top) and GSDMD processing (bottom) from transient expression of empty vector (EV), WT or mutant NLRP1-FL, or UPA-CARD in a reconstituted HEK293 inflammasome system, with and without addition of VbP. Anti-NLRP1-CT (FL and CT), anti-GSDMD, and anti-GAPDH antibodies were used in the immunoblots. *: non-specific bands; p30: GSDND N-terminal fragment from caspase-1 cleavage. b , Quantification of speck formation induced by expression of EV, WT or mutant NLRP1-FL, or UPA-CARD in the presence and absence of VbP. c , LDH release (top) and GSDMD processing (bottom) by direct expression of WT or mutant NLRP1 UPA-CARDs. CARD alone was also included. d , Quantification of speck formation induced by expression of WT or mutant NLRP1 UPA-CARD. e , A modelled UPA oligomer based on the near front-to-back interaction in the NLRP1-DPP9 ternary complex. In the model, the N-terminal tails of free UPAs are shown in either the UPA A or UPA B conformation in complex with DPP9, but in reality, this conformation is likely to be different. All data are representative of > 3 independent experiments. **** in (a) and (b): p < 0.0001 compared to EV by 2-way ANOVA with Tukey multiple comparison correction. *** in (c) and (d): p < 0.001 by two-sided Student’s t test.

Article Snippet: Primary antibodies used in this study include: DPP9 rabbit polyclonal Ab (Abcam, Ab42080), FLAG® M2 mouse monoclonal Ab (Sigma, F3165), GAPDH rabbit monoclonal Ab (Cell Signaling Technology, 14C10), NLRP1 mouse monoclonal Ab (R&D Systems, MAB6788), CASP1 rabbit polyclonal Ab (Cell Signaling Technology, 2225), GSDMDC1 rabbit polyclonal Ab (Novus, NBP2-33422), V5 rabbit polyclonal Ab (Abcam, Ab9116).

Techniques: Activity Assay, Expressing, Plasmid Preparation, Mutagenesis, Western Blot, Comparison

Journal: bioRxiv

Article Title: DPP9 directly sequesters the NLRP1 C-terminus to repress inflammasome activation

doi: 10.1101/2020.08.14.246132

Figure Lengend Snippet: a , Co-expression of GFP-ASC and NLRP1-FL (WT and mutants) or UPA-CARD (positive control). Autoactive constructs form GFP-ASC specks in the absence of VbP. Image quantitation is shown in . b , Enlarged GFP-ASC panels from (a). c , Co-expression of GFP-ASC and UPA-CARD (WT and interface III mutants). Image quantitation is shown in . d , Enlarged GFP-ASC panels from (c). All data are representative of > 3 independent experiments.

Article Snippet: Primary antibodies used in this study include: DPP9 rabbit polyclonal Ab (Abcam, Ab42080), FLAG® M2 mouse monoclonal Ab (Sigma, F3165), GAPDH rabbit monoclonal Ab (Cell Signaling Technology, 14C10), NLRP1 mouse monoclonal Ab (R&D Systems, MAB6788), CASP1 rabbit polyclonal Ab (Cell Signaling Technology, 2225), GSDMDC1 rabbit polyclonal Ab (Novus, NBP2-33422), V5 rabbit polyclonal Ab (Abcam, Ab9116).

Techniques: Expressing, Positive Control, Construct, Quantitation Assay

Journal: bioRxiv

Article Title: DPP9 directly sequesters the NLRP1 C-terminus to repress inflammasome activation

doi: 10.1101/2020.08.14.246132

Figure Lengend Snippet: a , Modelling of a FIIND polymer using the observed UPA A -UPA B relationship. Adjacent ZU5 molecules would clash, suggesting that UPA polymerization can only occur for free NLRP1-CTs. b , Modelled recruitment of free UPA at both UPA A and UPA B in the ternary complex with DPP9. The additional UPA subdomain next to FIND A clashes with the ZU5 subdomain, and the additional UPA next to UPA B clashes with both DPP9 monomers in the complex, suggesting that DPP9 inhibits UPA oligomerization.

Article Snippet: Primary antibodies used in this study include: DPP9 rabbit polyclonal Ab (Abcam, Ab42080), FLAG® M2 mouse monoclonal Ab (Sigma, F3165), GAPDH rabbit monoclonal Ab (Cell Signaling Technology, 14C10), NLRP1 mouse monoclonal Ab (R&D Systems, MAB6788), CASP1 rabbit polyclonal Ab (Cell Signaling Technology, 2225), GSDMDC1 rabbit polyclonal Ab (Novus, NBP2-33422), V5 rabbit polyclonal Ab (Abcam, Ab9116).

Techniques: Polymer

Journal: bioRxiv

Article Title: DPP9 directly sequesters the NLRP1 C-terminus to repress inflammasome activation

doi: 10.1101/2020.08.14.246132

Figure Lengend Snippet: a , Schematic representation of tertiary complex formation between the autocatalysis-deficient NLRP1-S1213A-FL, Ub-NLRP1-CT, and DPP9. Ub-NLRP1-CT is co- or post-translationally processed by endogenous deubiquitinases, resulting in NLRP1-CT with the native S1213 N-terminus. b , FLAG co-immunoprecipitation using FLAG-tagged WT NLRP1-FL or S1213A-FL and V5-tagged Ub-NLRP1-CT. Neither S1213A-FL (site A) nor NLRP1-CT (site B) alone pulled down endogenous DPP9. In contrast, co-expression of the two constructs formed a ternary complex with DPP9. c , Suppressed NLRP1-CT-induced cell death measured by LDH release in a reconstituted HEK293 inflammasome system when co-expressed with increasing amount of S1213A-FL. All data are representative of > 2 independent experiments. *: p < 0.05 and ***: p < 0.001 by two-sided Student’s t test. d , Schematic of NLRP1 activation and repression by DPP9. NLRP1-FL, together with DPP9, represses some threshold of free NLRP1-CTs. Enhanced degradation of NLRP1-FL, or displacement of the NLRP1-CT, leads to inflammasome signalling.

Article Snippet: Primary antibodies used in this study include: DPP9 rabbit polyclonal Ab (Abcam, Ab42080), FLAG® M2 mouse monoclonal Ab (Sigma, F3165), GAPDH rabbit monoclonal Ab (Cell Signaling Technology, 14C10), NLRP1 mouse monoclonal Ab (R&D Systems, MAB6788), CASP1 rabbit polyclonal Ab (Cell Signaling Technology, 2225), GSDMDC1 rabbit polyclonal Ab (Novus, NBP2-33422), V5 rabbit polyclonal Ab (Abcam, Ab9116).

Techniques: Immunoprecipitation, Expressing, Construct, Activation Assay

Journal: PLoS ONE

Article Title: Transcription Factor ATF4 Induces NLRP1 Inflammasome Expression during Endoplasmic Reticulum Stress

doi: 10.1371/journal.pone.0130635

Figure Lengend Snippet: (A) Un-differentiated THP-1 cells were treated with the indicated stimuli for 6 hours. NLRP1 levels were measured by quantitative real-time PCR (qPCR) using cyclophillin A as an endogenous control. Semi-quantitative RT-PCR using a different NLRP1 primer set and GAPDH as a control is also shown. (B) HeLa cells were treated either with BFA or TG for the indicated times. NLRP1 mRNA levels were measured by qPCR and RT-PCR. Spliced and un-spliced XBP-1 forms were also evaluated by RT-PCR. (C) HCT116 cells were treated with the indicated stimuli for 24 hours. NLRP1 and NOD1 mRNA levels were measured by qPCR. (D) Cell lysates from wild-type or NLRP1 −/− HeLa, THP-1 and K562 cells, untreated or treated with BFA for 20 hours, were normalized for total protein content. Cell extracts were then subjected to SDS-PAGE/immunoblot analysis before and after immunoprecipitation with NLRP1 antibody. Vinculin was detected as loading control. NLRP1 mRNA levels were also measured by RT-PCR. Each panel is representative of at least three independent experiments. (DMSO: dimethyl sulfoxide, TM: tunicamycin, TG: thapsigargin, MSU: monosodium urate crystals, BFA: brefeldin A, PolyI:C: polyinosinic-polycytidylic acid, FLA: flagellin, MDP: muramyl dipeptide, R837: Imiquimod)

Article Snippet: NLRP1 antibody (AF6788) was from R&D Systems.

Techniques: Real-time Polymerase Chain Reaction, Control, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, SDS Page, Western Blot, Immunoprecipitation

Journal: PLoS ONE

Article Title: Transcription Factor ATF4 Induces NLRP1 Inflammasome Expression during Endoplasmic Reticulum Stress

doi: 10.1371/journal.pone.0130635

Figure Lengend Snippet: (A) HeLa cells were infected with increasing concentrations of murine Xbp-1s and Atf4 adenovirus for 24 hours and NLRP1 mRNA was measured by qPCR. (B) IRE1α, PERK, ATF6 and XBP-1s were down-regulated using siRNA in HeLa cells. Cells were treated with BFA for 20 hours and mRNA levels were measured by qPCR. IRE1α, PERK, ATF6 and XBP-1s knock-down was verified by SDS-PAGE/immunoblotting. Each panel is representative of at least three independent experiments.

Article Snippet: NLRP1 antibody (AF6788) was from R&D Systems.

Techniques: Infection, Knockdown, SDS Page, Western Blot

Journal: PLoS ONE

Article Title: Transcription Factor ATF4 Induces NLRP1 Inflammasome Expression during Endoplasmic Reticulum Stress

doi: 10.1371/journal.pone.0130635

Figure Lengend Snippet: (A) Different NLRP1 promoter regions were cloned into a luciferase reporter vector. At 24 hours after transfection, HeLa cells were treated with BFA for 20 hours before measuring luciferase activity. (B) HeLa cells were transfected with wild-type NLRP1 promoter or a version carrying mutations in the ATF4-binding motif. At 24 hours after transfection, cells were treated with BFA for 20 hours before measuring luciferase activity. (C) The indicated NLRP1 promoter-luciferase vectors were transfected into either wild-type or ATF4 −/− HeLa cells. At 24 hours after transfection, cells were treated with BFA for 20 hours before measuring luciferase activity. (D) ChIP analysis of un-stimulated (DMSO) or BFA-stimulated HeLa cells, followed by qPCR analysis of ATF4 occupancy at the NLRP1 and ATF3 promoter. ATF3 was used as positive control. Each panel is representative of at least three independent experiments.

Article Snippet: NLRP1 antibody (AF6788) was from R&D Systems.

Techniques: Clone Assay, Luciferase, Plasmid Preparation, Transfection, Activity Assay, Binding Assay, Positive Control

Journal: EMBO Molecular Medicine

Article Title: TKI-mediated inhibition of NLRP1 inflammasome restores erythropoiesis in DBA syndrome

doi: 10.1038/s44321-025-00368-3

Figure Lengend Snippet: ( A ) Chronogram of the experiment. ( B – D ) K562 cells were pretreated with 0.1 μM nilotinib ( B – E ), 0.1–1 μM imatinib and 0.1–1 μM dasatinib ( B , C ), 20–1000 nM ponatinib and 100–1000 bosutinib ( D ) for 24 h, and then differentiated with 50 μM hemin for another 24 h. Hemoglobin accumulation ( A – C ) and NLRP1, phosphorylated P38, phosphorylated JNK1/JNK2, GATA1, ZAKα and ACTB/GAPDH ( A – D ) amounts were then evaluated by Western blot. HEK293T cells were treated for 24 h with 1 µM anisomycin ( A ) in either the presence or absence of 0.1 µM nilotinib as a control of JNK1/JNK2 activation ( C ). Immunoblots are representative of three independent experiments. For ( B ) and ( D ), GATA1 and ACTB were detected on the same membrane after stripping and reprobing, whereas the remaining proteins were analyzed on separate membranes. *, GATA1. ( D ) CASP1 activity was measured by FLICA-CASP1 then analyzed by flow cytometry. Representative dot plots at different differentiation times are shown. .

Article Snippet: The primary antibodies used were human GATA1 (1:1000, #3535, Cell Signaling), human NLRP1 (1:1000 #AF6788, R&D Systems), human ZAKα (1:1000, #A301-993A, Bethyl Laboratories), human phosphoP38 (1:1000, #MA5-15177, ThermoFisher Scientific), pJNK1/pJNK2 (1:1000 #MA5-15228, Invitrogen), GAPDH (1:2500, #ab181602, Abcam), ACTB-HRP (1:5000, #sc-47778, Santa Cruz Biotechnology).

Techniques: Western Blot, Control, Activation Assay, Membrane, Stripping Membranes, Activity Assay, Flow Cytometry

Journal: Redox Biology

Article Title: Ubiquitination as a key regulatory mechanism for O 3 -induced cutaneous redox inflammasome activation

doi: 10.1016/j.redox.2022.102440

Figure Lengend Snippet: O 3 activated NLRP1 inflammasome via Caspase I (a) Immunofluorescence staining for NLRP1 (green) and ASC (red) in HaCaT cells silenced for NLRP1 10 nM for 24 h and exposed to O 3 . The blue staining (DAPI) represents nuclei. Images were taken at 40 × magnification (scale bar = 40 μm) and the fluorescent signal was quantified using ImageJ software. (b) Protein expression levels and relative quantification graph of p20 Caspase 1 over pro-Caspase 1 in HaCaT cells silenced for NLRP1 10 nM and then exposed to O 3 . (c) IL-1β released levels in media of HaCaT cells silenced for NLRP1 10 nM for 24 h and then exposed to O 3 . (d) Protein expression levels of p20 Caspase 1 over pro-Caspase 1 in HaCaT cells pre-treated with Caspase 1 inhibitor Z-YVAD-fmk 2 and 10 μM for 1 h and then exposed to O 3 . IL-1β mRNA expression levels ( e ) and released levels of IL-1β ( f ) in HaCaT cells pre-treated with Caspase 1 inhibitor Z-YVAD-fmk 2 μM for 1 h and then exposed to O 3 . For all the experiments HaCaT cells were exposed to 0.4 ppm O 3 for 1 h and then collected at the indicated timepoints post 1-h O 3 exposure. For all western blotting the protein expression level was quantified using ImageJ software and β-actin was used as internal control. Data are the results of the averages of at least three different experiments, *p < 0.05 and # p < 0.05 by 2-way ANOVA followed by Tukey’s post-hoc comparison test. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: HaCaT Cells and human 1° KC were transfected with siRNA against human NLRP1 by using the NLRP1 (NLRP1) Human siRNA Oligo Duplex , Locus ID 22861, sequence SR307826B (Cat. SR307826 , Origene TM Technologies , Rockville, MD, USA) or the negative control Scrambled (Scrbl) siRNA using the Lipofectamine RNAiMAX transfection reagent (Cat. 13778030, Invitrogen, ThermoFisher Scientific, Waltham, MA, USA), according to the datasheet.

Techniques: Immunofluorescence, Staining, Software, Expressing, Quantitative Proteomics, Western Blot, Control, Comparison

Journal: Redox Biology

Article Title: Ubiquitination as a key regulatory mechanism for O 3 -induced cutaneous redox inflammasome activation

doi: 10.1016/j.redox.2022.102440

Figure Lengend Snippet: NLRP1 inflammasome activation by H 2 O 2 (a) H 2 O 2 levels production in media of HaCaT cells pre-treated with 1000 U/ml of Catalase (Cat) for 2 h and then exposed to O 3 assessed by AmplexRed assay. (b) mRNA expression levels of NLRP1 (upper panel) and ASC (bottom panel) in HaCaT cells pre-treated with 1000 U/ml of Cat for 2 h and then exposed to O 3. (c) Double Immunofluorescence staining for NLRP1 (red) and ASC (green) in HaCaT cells pre-treated with Cat 1000 U/ml for 2 h and then exposed to O 3. Blue staining (DAPI) represents nuclei. Images were taken at 100 × magnification (scale bar = 2.5 μm); the fluorescent levels were quantified using ImageJ software. (d) Caspase 1 released levels in media of HaCaT cells pre-treated with Cat 1000 U/ml for 2 h and then exposed to O 3 . (e) IL-1β mRNA expression levels in HaCaT cells pre-treated with Cat 1000 U/ml for 2 h and then exposed to O 3. (f) Released levels of IL-1β in media of HaCaT cells pre-treated with Cat 1000 U/ml for 2 h and then exposed to O 3 . For all the experiments HaCaT cells were exposed to 0.4 ppm O 3 for 1 h. Samples were collected at the indicated timepoints post 1-h O 3 exposure. For all western blotting the protein expression level was quantified using ImageJ software and β-actin was used as internal control. Data are the results of the averages of at least three different experiments, *p < 0.05 and # p < 0.05 by 2-way ANOVA followed by Tukey’s post-hoc comparison test. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: HaCaT Cells and human 1° KC were transfected with siRNA against human NLRP1 by using the NLRP1 (NLRP1) Human siRNA Oligo Duplex , Locus ID 22861, sequence SR307826B (Cat. SR307826 , Origene TM Technologies , Rockville, MD, USA) or the negative control Scrambled (Scrbl) siRNA using the Lipofectamine RNAiMAX transfection reagent (Cat. 13778030, Invitrogen, ThermoFisher Scientific, Waltham, MA, USA), according to the datasheet.

Techniques: Activation Assay, Expressing, Double Immunofluorescence Staining, Staining, Software, Western Blot, Control, Comparison

Journal: Redox Biology

Article Title: Ubiquitination as a key regulatory mechanism for O 3 -induced cutaneous redox inflammasome activation

doi: 10.1016/j.redox.2022.102440

Figure Lengend Snippet: Involvement of 4HNE protein adducts in NLRP1 inflammasome activation (a) 4HNE protein expression levels in HaCaT cells exposed at the indicated doses of O 3 for 1 h and collected right after the end of O 3 exposure. (b) Immunoprecipitation assay for NLRP1 probed with 4HNE in HaCaT cells pre-treated with proteasome inhibitor MG-132 20 μM for 2 h and exposed to O 3 or H 2 O 2 50 μM for 1 h. Samples were collected right after the end of 1-h O 3 /H 2 O 2 exposures. (c) Double Immunofluorescence staining for NLRP1 (red) and 4HNE (green) in HaCaT cells after O 3 exposure. The blue staining (DAPI) represents nuclei. Images were taken at 60 × magnification (scale bar = 10 μm) and the fluorescent levels were quantified using ImageJ software. (d) Quantification of protein expression levels of ubiquitinated proteins in HaCaT cells exposed to O 3 . (e) Protein expression levels of ubiquitinated-NLRP1 after Immunoprecipitation for NLRP1 in HaCaT cells pre-treated with proteasome inhibitor MG-132 20 μM for 2 h and Cat 1000 U/ml for 2 h and exposed to O 3 right after the end of exposure. For all the experiments HaCaT cells were exposed to 0.4 ppm O 3 for 1 h and then collected at the indicated timepoints post 1-h O 3 exposure. For all western blotting the protein expression level was quantified using ImageJ software and β-actin was used as internal control. Data are the results of the averages of at least three different experiments, *p < 0.05 and # p < 0.05 by 2-way ANOVA followed by Tukey’s post-hoc comparison test. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: HaCaT Cells and human 1° KC were transfected with siRNA against human NLRP1 by using the NLRP1 (NLRP1) Human siRNA Oligo Duplex , Locus ID 22861, sequence SR307826B (Cat. SR307826 , Origene TM Technologies , Rockville, MD, USA) or the negative control Scrambled (Scrbl) siRNA using the Lipofectamine RNAiMAX transfection reagent (Cat. 13778030, Invitrogen, ThermoFisher Scientific, Waltham, MA, USA), according to the datasheet.

Techniques: Activation Assay, Expressing, Immunoprecipitation, Double Immunofluorescence Staining, Staining, Software, Western Blot, Control, Comparison

Journal: Redox Biology

Article Title: Ubiquitination as a key regulatory mechanism for O 3 -induced cutaneous redox inflammasome activation

doi: 10.1016/j.redox.2022.102440

Figure Lengend Snippet: NLRP1 activation is mediated by its ubiquitination. Protein expression levels of NLRP1 (a) in HaCaT cells pre-treated or not with the proteasome inhibitor MG-132 and then exposed to O 3 . (b) Double immunofluorescence staining for NLRP1 (green) and ASC (red), in HaCaT cells pre-treated with MG-132 and then exposed to O 3 . Blue staining (DAPI) represents nuclei. Images were taken at 40 × magnification (scale bar = 20 μm). The fluorescent levels were quantified using ImageJ software. Protein expression levels of p20 Caspase 1 over Pro-Caspase 1 ( c ), and UBR2 ( d ) in HaCaT cells pre-treated with MG-132 and exposed to O 3 . (e) Double IF staining for NLRP1 (red) and UBR2 (green) in HaCaT cells pre-treated with MG-132 and exposed to O 3 . Blue staining (DAPI) represents nuclei. Images were taken at 40 × magnification (scale bar = 20 μm). For all the experiments HaCaT cells were pre-treated or not with the proteasome inhibitor MG-132 20 μM for 2 h and then exposed to O 3 0.4 ppm for 1 h. Samples were collected at the indicated timepoints post 1-h O 3 exposure. For all western blotting the protein expression level was quantified using ImageJ software and β-actin or Red ponceau were used as internal control. Data are the results of the averages of at least three different experiments, *p < 0.05 and # p < 0.05 by 2-way ANOVA followed by Tukey’s post-hoc comparison test. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: HaCaT Cells and human 1° KC were transfected with siRNA against human NLRP1 by using the NLRP1 (NLRP1) Human siRNA Oligo Duplex , Locus ID 22861, sequence SR307826B (Cat. SR307826 , Origene TM Technologies , Rockville, MD, USA) or the negative control Scrambled (Scrbl) siRNA using the Lipofectamine RNAiMAX transfection reagent (Cat. 13778030, Invitrogen, ThermoFisher Scientific, Waltham, MA, USA), according to the datasheet.

Techniques: Activation Assay, Ubiquitin Proteomics, Expressing, Double Immunofluorescence Staining, Staining, Software, Western Blot, Control, Comparison

Journal: Redox Biology

Article Title: Ubiquitination as a key regulatory mechanism for O 3 -induced cutaneous redox inflammasome activation

doi: 10.1016/j.redox.2022.102440

Figure Lengend Snippet:

Article Snippet: HaCaT Cells and human 1° KC were transfected with siRNA against human NLRP1 by using the NLRP1 (NLRP1) Human siRNA Oligo Duplex , Locus ID 22861, sequence SR307826B (Cat. SR307826 , Origene TM Technologies , Rockville, MD, USA) or the negative control Scrambled (Scrbl) siRNA using the Lipofectamine RNAiMAX transfection reagent (Cat. 13778030, Invitrogen, ThermoFisher Scientific, Waltham, MA, USA), according to the datasheet.

Techniques: Sequencing

Journal: Molecular cell

Article Title: Human NLRP1 is a sensor of pathogenic coronavirus 3CL proteases in lung epithelial cells.

doi: 10.1016/j.molcel.2022.04.033

Figure Lengend Snippet: Figure 1. NLRP1 is an innate immune sensor of SARS-CoV-2 infection (A) Western blot examination of NLRP1, NLRP3, and ACTIN in various human epithelial cells and cell lines. (B) Cell death (LDH) and viability (ATP Glo) evaluation in A549ACE2/NLRP1+ and A549ACE2/NLRP1 airway epithelial cell lines infected with various multiplicity of infec- tion (MOI) of SARS-CoV-2 for 24 h. (C) Florescence microscopy and associated quantifications of ASC-GFP specks in A549ACE2/NLRP1+/ASC-GFP and A549ACE2/NLRP1/ASC-GFP airway epithelial cell lines infected with SARS-CoV-2 (MOI 0.05) for 24 h. Nucleus was stained with Hoechst (blue), and nucleocapsid (N) was stained in red after fixation. Brightfield/ASC-GFP pictures were taken in dish during cell infection. Images shown are from one experiment and are representative of n = 3 independent

Article Snippet: In vitro cleavage assays Recombinant proteins The human purified recombinant gasdermin D (6 mM final concentration), produced and purified in the lab or the human recombinant NLRP1 (0.7 mM final concentration), from Origen (CAT#: TP316481) was incubated in 20 mM Tris at pH7,5 150 mM NaCl 1 mM DTT with recombinant SARS-CoV-2 3CL protease (Sigma-Aldrich SAE0172, R&D Systems, Novus Biologicals), recombinant SARS-CoV2 PLpro recombinant (Sigma-Aldrich) SARS-CoV-1 or MERS-CoV 3CL (Novus Biologicals) at molar ratio of 3:1 otherwise as indicated in figure legends, for O/N at 4 C, in presence or absence of protease inhibitor (PF-00835231, 10 mM – Selleck Chemicals S9731).

Techniques: Infection, Western Blot, Microscopy, Staining

Journal: Molecular cell

Article Title: Human NLRP1 is a sensor of pathogenic coronavirus 3CL proteases in lung epithelial cells.

doi: 10.1016/j.molcel.2022.04.033

Figure Lengend Snippet: Figure 2. SARS-CoV-2 NSP5 protease-cleaved NLRP1 at the Q333 site nucleates NLRP1 inflammasome (A) Florescence microscopy and associated quantifications of ASC-GFP specks in A549NLRP1+/ASC-GFP and A549NLRP1/ASC-GFP airway epithelial cell lines in- fected with SARS-CoV-2 (MOI 0.05) for 24 h in the presence or absence of proteasome inhibitor bortezomib (0.1 mM) or inhibitor of the glycine N-degron pathway MLN4924 (1 mM). Images shown are from one experiment and are representative of n = 3 independent experiments; scale barss 10 mm. For quantifications, the percentage of cells with ASC complexes was determined by determining the ratios of cells positives for ASC speckles on the total cells presents in the wells. At least 10 fields from n = 3 independent experiments were analyzed. Values are expressed as mean ± SEM. (B) Western blot examination of NLRP1 cleavage using an anti-NLRP1 N-terminal antibody (aa 1–323) upon coincubation of SARS-CoV-2, SARS-CoV-1, or MERS-CoV 3CL (NSP5) proteases (5 mM) with A549NLRP1+ airway epithelial cell lysates in presence or absence of the 3CL inhibitors GC-376 (10 mM) or PF- 00835231 (10 mM). NLRP1 N-terminal, NLRP1 C-terminal, NSP5, and ACTIN were immunoblotted. (C) Florescence microscopy and associated quantifications of ASC-GFP specks in A549NLRP1+/ASC-GFP airway epithelial cell lines transduced with a doxycycline (dox)-inducible plasmid encoding NSP5 or its catalytically inactive mutant NSP5C145A. Images shown are from one experiment and are representative of n = 3 independent experiments; scale bars, 10 mm. For quantifications, the percentage of cells with ASC complexes was determined by determining the ratios of cells positives for ASC speckles on the total cells presents in the wells. At least 10 fields from n = 3 independent experiments were analyzed. Values are expressed as mean ± SEM. (D) Schematic representation of the approximate NLRP1 N-terminal fragment generated by NSP5 protease cut. (E) Western blot examinations of the ability of NSP5 to cleave various NLRP1 constructs mutated in glutamine (Q) at various sites. Immunoblots show anti-N-ter- minal NLRP1, ACTIN, and NSP5. (F) Florescence microscopy and associated quantifications of ASC-GFP specks in A549NLRP1+/ASC-GFP or A549NLRP1Q333A/ASC-GFP airway epithelial cell lines transduced with a doxycycline (dox)-inducible plasmid encoding NSP5 or its catalytically inactive mutant NSP5C145A. Images shown are from one experiment

Article Snippet: In vitro cleavage assays Recombinant proteins The human purified recombinant gasdermin D (6 mM final concentration), produced and purified in the lab or the human recombinant NLRP1 (0.7 mM final concentration), from Origen (CAT#: TP316481) was incubated in 20 mM Tris at pH7,5 150 mM NaCl 1 mM DTT with recombinant SARS-CoV-2 3CL protease (Sigma-Aldrich SAE0172, R&D Systems, Novus Biologicals), recombinant SARS-CoV2 PLpro recombinant (Sigma-Aldrich) SARS-CoV-1 or MERS-CoV 3CL (Novus Biologicals) at molar ratio of 3:1 otherwise as indicated in figure legends, for O/N at 4 C, in presence or absence of protease inhibitor (PF-00835231, 10 mM – Selleck Chemicals S9731).

Techniques: Microscopy, Western Blot, Transduction, Plasmid Preparation, Mutagenesis, Generated, Construct

Journal: Clinical & Translational Immunology

Article Title: A novel dual NLRP1 and NLRP3 inflammasome inhibitor for the treatment of inflammatory diseases

doi: 10.1002/cti2.1455

Figure Lengend Snippet: ADS032 is a novel NLRP3 inhibitor. (a) ADS032 structure. (b) Immortalised bone marrow‐derived macrophages (iBMDMs) were seeded at 4 × 10 5 mL −1 prior to priming with LPS (100 ng mL −1 ) for 3 h. Media were removed 60 min prior to challenge and replaced with serum‐free media containing ADS032 (3.5–350 μ m ) as indicated, or DMSO control (0.8% v/v). Macrophages were stimulated with either nigericin (6 μ m ) or silica (250 μg mL −1 ) as indicated for 120 or 360 min respectively. Cellular supernatants were analysed for secreted IL‐1β, TNF‐α by ELISA or cell death by LDH assay as indicated. (c) iBMDMs were treated with a range of ADS032 concentrations for 60 min in serum‐free media prior to challenge with nigericin for 120 min. The results shown are representative of three independent experiments carried out in triplicate and presented as the mean ± SEM. Non‐linear regression analysis was performed, and the curve of the log [ M ] ADS032 versus the normalised response (variable slope) is presented. (d) LPS (3 h;100 ng mL −1 )‐primed iBMDMs were pre‐treated with ADS032 (20 and 100 μg mL −1 ) or MCC950 (MCC: 5 μg mL −1 ) in serum‐free media for 60 min and treated with NLRP3 (nigericin, 6 μ m , 120 min; monosodium urate (MSU), 250 μg mL −1 , 6 h; and silica, 250 μg mL −1 ; 6 h), NLRP1 (L18‐MDP, 100 μg mL −1 , 16 h), non‐canonical NLRP3 (transfected LPS Escherichia coli serotype 0111 B4, 2.0 μg mL −1 , 16 h), AIM2 (poly dA:dT, 1 μg mL −1 , 8 h) and NLRC4 (flagellin, 20 μg mL −1 , 16 h) agonists as indicated. Secreted IL‐1β was assayed by ELISA and the results shown are representative of three independent experiments carried out in triplicate and presented as the mean ± SEM. (e) IC 50 value of L18‐MDP (NLRP1) activity was determined as normalised IL‐1β secretion in ADS032‐treated iBMDMs. (f) BMDMs generated from wild‐type mice, and (g) PMA‐differentiated THP‐1 macrophages were primed with LPS for 3 h, treated with ADS032 (100 and 300 μ m ) or MCC950 (5 μ m ) where indicated for 60 min, then subsequently challenged with nigericin for 180 min. Cultured supernatants were assayed for secreted IL‐1β and caspase‐1 following 4–12% SDS‐PAGE, transfer to PVDF membrane and immunoblotting (IB) with indicated antibodies. The results shown are representative of three independent experiments.

Article Snippet: Recombinant full‐length human NLRP1 (Creative Biomart, Shirley, USA) or human NLRP3 ΔLRR 1–536 (Cusabio, Houston, USA) (2 μg) were resuspended in lysis buffer (50 m m Tris–HCl pH 7.4, 150 m m NaCl and 0.5% Igepal CA‐630) and incubated with ADS165 or MCC950 where indicated (total volume 40 μL) for 20 min prior to photo‐crosslinking with a UVA fluorescent light box at 365 nm (AnalytikJena, Jena, Germany) for 20 min. ADS165 was then added for a further 20 min and cross‐linked for 20 min.

Techniques: Derivative Assay, Control, Enzyme-linked Immunosorbent Assay, Lactate Dehydrogenase Assay, Transfection, Activity Assay, Generated, Cell Culture, SDS Page, Membrane, Western Blot

Journal: Clinical & Translational Immunology

Article Title: A novel dual NLRP1 and NLRP3 inflammasome inhibitor for the treatment of inflammatory diseases

doi: 10.1002/cti2.1455

Figure Lengend Snippet: ADS032 directly binds to NLRP1 and NLRP3. (a, b) ADS165 and ADS167 structure. (c) Recombinant NLRP3 (2 μg) or (e) NLRP1 (2 μg) was co‐incubated where indicated with ADS167 or MCC950 for 20 min and then irradiated with UV 365 nm for a further 20 min. ADS165 was then co‐incubated for 20 min and UV treated for 20 min to cross‐link‐associated compound and protein. Protein was separated by SDS‐PAGE and immunoblotted (IB) with anti‐PEG to visualise ADS165‐linked protein. (d) iBMDM cells stably expressing NLRP3‐Flag were treated for 30 min with ADS165, ADS167 (1 m m ) or MCC950 (50 μ m ) for 30 min, and irradiated with UV 365 nm for 30 min. Cells were lysed with buffer and immunoprecipitation (IP) of NLRP3 was performed with α‐NLRP3 (D4D8T) antibody. Levels of precipitated (IP) and total cellular lysate (Input) NLRP3 were determined by immunoblotting with α‐NLRP3 (Cryo‐2) antibody. (f) NLRP1‐Flag was expressed in HEK293T cells (1 × 10 6 ) for 20 h and treated with ADS165 (1 m m ) for 30 min where indicated, followed by UV 365 nm for a further 30 min. Cells were lysed with lysis buffer and NLRP1 immunoprecipitated with α‐Flag (M2) antibody. Proteins were separated by SDS‐PAGE and ADS165‐linked NLRP1 identified by immunoblot with α‐PEG, while total precipitated NLRP1 was visualised by immunoblot with α‐NLRP1 and total cell lysate by α‐Flag. The results shown are representative of three independent experiments.

Article Snippet: Recombinant full‐length human NLRP1 (Creative Biomart, Shirley, USA) or human NLRP3 ΔLRR 1–536 (Cusabio, Houston, USA) (2 μg) were resuspended in lysis buffer (50 m m Tris–HCl pH 7.4, 150 m m NaCl and 0.5% Igepal CA‐630) and incubated with ADS165 or MCC950 where indicated (total volume 40 μL) for 20 min prior to photo‐crosslinking with a UVA fluorescent light box at 365 nm (AnalytikJena, Jena, Germany) for 20 min. ADS165 was then added for a further 20 min and cross‐linked for 20 min.

Techniques: Recombinant, Incubation, Irradiation, SDS Page, Stable Transfection, Expressing, Immunoprecipitation, Western Blot, Lysis

Journal: Clinical & Translational Immunology

Article Title: A novel dual NLRP1 and NLRP3 inflammasome inhibitor for the treatment of inflammatory diseases

doi: 10.1002/cti2.1455

Figure Lengend Snippet: ADS032 effectively inhibits NLRP1 and NLRP3 inflammasome activity in human macrophages and bronchial epithelial cells. PMA‐differentiated THP‐1 cells primed with LPS (100 ng mL −1 ) for 3 h, pre‐treated or not with ADS032 (35–350 μ m ) for 60 min and then challenged with (a) NLRP3 agonists nigericin (6 μ m ) or silica (250 μg mL −1 ) for 120 or 360 min, respectively, or (c) NLPR1 agonists L18‐MDP (100 μg mL −1 ) or transfected poly I:C (200 ng mL −1 ) for 16 or 8 h respectively. (b) Human monocyte‐derived macrophages were treated for 3 h with 50 pg mL −1 LPS, treated with ADS032 (20–350 μ m ) or MCC950 (MCC; 5 μ m ) in serum‐free media for 60 min, then challenged with nigericin (6 μ m ) for a further 120 min. Bronchial epithelial cells obtained from normal patients were treated with ADS032 as indicated for 60 min and (d) treated with poly I:C (200 ng mL −1 ) for a further 8 h or (e) talabostat (2.5 μ m ; 24 h) and anisomycin (2.0 μ m ; 8 h). Cultured supernatants were assayed for secreted IL‐1β by ELISA. The results shown are the pooled data of three independent experiments carried out in triplicate and presented as the mean ± SEM.

Article Snippet: Recombinant full‐length human NLRP1 (Creative Biomart, Shirley, USA) or human NLRP3 ΔLRR 1–536 (Cusabio, Houston, USA) (2 μg) were resuspended in lysis buffer (50 m m Tris–HCl pH 7.4, 150 m m NaCl and 0.5% Igepal CA‐630) and incubated with ADS165 or MCC950 where indicated (total volume 40 μL) for 20 min prior to photo‐crosslinking with a UVA fluorescent light box at 365 nm (AnalytikJena, Jena, Germany) for 20 min. ADS165 was then added for a further 20 min and cross‐linked for 20 min.

Techniques: Activity Assay, Transfection, Derivative Assay, Cell Culture, Enzyme-linked Immunosorbent Assay