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A–D (A, B) Human IL‐1β (hIL‐1β) concentrations in cell‐free supernatants of LPS‐primed (10 ng ml −1 , 150 min) primary human macrophages that were left untreated, or pre‐incubated with VHH ASC or mutVHH ASC (100 µg ml −1 ), <t>CRID3</t> (50 µM) or VX‐765 (50 µM) for 30 min before stimulation with (A) nigericin (10 µM), or (B) PFO (30 ng ml −1 ) for 2 h. (C‐D) Mouse IL‐1β (mIL‐1β) concentrations in cell‐free supernatants of LPS‐primed mouse BMDMs (200 ng ml −1 , 150 min), incubated with VHHs, CRID3 or VX765, before activation with nigericin (10 µM), or PFO (250 ng ml −1 ). Data is combined from two independent experiments, each performed with two donors (A, B) or mice (C, D), represented with individual symbols (4 donors or mice in total). Data is displayed as floating bars with the max/min values and mean (thicker band). E, F (E) Epifluorescence microscopy imaging and (F) quantification of ASC speck formation in BMDMs from ASC‐mCitrine (Green) transgenic mice. Cells were primed with LPS (200 ng ml −1 , 150 min), pre‐treated with VX‐765 (50 µM, 30 min), then treated with VHH ASC , VHH mASC (100 µg ml −1 ) or CRID3 (50 µM) for another 30 min before stimulation with nigericin (top), or PFO (bottom) for 2 h and finally fixed with 4% PFA. Nuclei was stained with DRAQ5 (Blue). Scale bars: 100 μm. Images in (E) are from one representative out of three independent experiments that were quantified in F. Data in F is displayed as floating bars with the max/min values and mean (thicker band). Data information: ** P < 0.005; *** P < 0.0002; **** P < 0.0001, One‐way ANOVA, multiple comparison (Tukey test).

Crid3, supplied by Tocris, used in various techniques. Bioz Stars score: 94/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Nanobodies dismantle post‐pyroptotic ASC specks and counteract inflammation in vivo"

Article Title: Nanobodies dismantle post‐pyroptotic ASC specks and counteract inflammation in vivo

Journal: EMBO Molecular Medicine

doi: 10.15252/emmm.202115415

Figure Legend Snippet: A–D (A, B) Human IL‐1β (hIL‐1β) concentrations in cell‐free supernatants of LPS‐primed (10 ng ml −1 , 150 min) primary human macrophages that were left untreated, or pre‐incubated with VHH ASC or mutVHH ASC (100 µg ml −1 ), CRID3 (50 µM) or VX‐765 (50 µM) for 30 min before stimulation with (A) nigericin (10 µM), or (B) PFO (30 ng ml −1 ) for 2 h. (C‐D) Mouse IL‐1β (mIL‐1β) concentrations in cell‐free supernatants of LPS‐primed mouse BMDMs (200 ng ml −1 , 150 min), incubated with VHHs, CRID3 or VX765, before activation with nigericin (10 µM), or PFO (250 ng ml −1 ). Data is combined from two independent experiments, each performed with two donors (A, B) or mice (C, D), represented with individual symbols (4 donors or mice in total). Data is displayed as floating bars with the max/min values and mean (thicker band). E, F (E) Epifluorescence microscopy imaging and (F) quantification of ASC speck formation in BMDMs from ASC‐mCitrine (Green) transgenic mice. Cells were primed with LPS (200 ng ml −1 , 150 min), pre‐treated with VX‐765 (50 µM, 30 min), then treated with VHH ASC , VHH mASC (100 µg ml −1 ) or CRID3 (50 µM) for another 30 min before stimulation with nigericin (top), or PFO (bottom) for 2 h and finally fixed with 4% PFA. Nuclei was stained with DRAQ5 (Blue). Scale bars: 100 μm. Images in (E) are from one representative out of three independent experiments that were quantified in F. Data in F is displayed as floating bars with the max/min values and mean (thicker band). Data information: ** P < 0.005; *** P < 0.0002; **** P < 0.0001, One‐way ANOVA, multiple comparison (Tukey test).

Techniques Used: Incubation, Activation Assay, Epifluorescence Microscopy, Imaging, Transgenic Assay, Staining, Comparison

A, B Cell viability (CTB) assay on LPS‐primed primary human macrophages that were left untreated, or pre‐incubated with VHH ASC or mutVHH ASC (100 µg ml −1 ), CRID3 (50 µM) or VX‐765 (50 µM) for 30 min before being activated with (A) nigericin (10 µM), or (B) PFO (30 ng ml −1 ) for 2 h. Data is from the experiments displayed in Fig A and B. C Human IL‐1β (hIL‐1β) concentrations in cell‐free supernatants (left), and cell viability assay (right) of LPS‐primed primary human macrophages that were incubated with VHH ASC or mutVHH ASC (100 µg ml −1 ), CRID3 (50 µM) or VX‐765 (50 µM) for 30 min before being stimulated with 2.5 mM ATP. D hIL‐1β concentrations in cell‐free supernatants (top), and cell viability assay (bottom) of PMA‐differentiated THP‐1 cells treated with VHH ASC or mutVHH ASC (100 µg ml −1 ), CRID3 (10 µM) or VX‐765 (50 µM) for 30 min before 4.5 h stimulation with 250 µg ml −1 MSU crystals. E hIL‐1β concentrations in cell‐free supernatants (top), and cell viability assay (bottom) of LPS‐primed primary human macrophages that were incubated with VHH ASC or mutVHH ASC (100 µg ml −1 ), CRID3 (50 µM) or VX‐765 (50 µM) for 30 min before being stimulated with 0.1 µg ml −1 /0.5 µg ml −1 mixture of LFn‐BsaK and PA for 2 h. F hIL‐1β concentrations in cell‐free supernatants (top), and cell viability assay (bottom) of Pam3CysK4‐primed (1 µg ml −1 ) primary human CD14 + monocytes that were incubated with VHH ASC or mutVHH ASC (100 µg ml −1 ), or VX‐765 (50 µM) for 30 min before being stimulated with 1 µg ml −1 TcdA. G hIL‐1β concentrations in cell‐free supernatants (top), and cell viability assay (bottom) of keratinocyte cells (N‐TERT) that were treated with VHH ASC or mutVHH ASC (100 µg ml −1 ), or VX‐765 (50 µM), then directly stimulated with 30 µM Val‐boroPro (VbP) for 22 h. H hIL‐1β concentrations in cell‐free supernatants (top), and cell viability assay (bottom) of PMA‐differentiated THP‐1 cells treated with IFNγ (500 U ml −1 ) for 16 h and that were incubated with VHH ASC or mutVHH ASC (100 µg ml −1 ), CRID3 (10 µM) or VX‐765 (50 µM) for 30 min before 2 h stimulation with 1 µg ml −1 poly(dA:dT) in complex with Lipofectamine 2000. Data information: Data is representative of either two independent experiments, each run with one to two donors (A–C, E, F, 3 or 4 donors in total) or at least 3 independent experiments (D, G, H). Each symbol represents one donor or independent experiment. ns P > 0.05; * P < 0.05; ** P < 0.005; *** P < 0.0002; **** P < 0.0001, One‐way ANOVA, multiple comparison (Tukey test). Data is displayed as floating bars with the max/min values and mean (thicker band).

Figure Legend Snippet: A, B Cell viability (CTB) assay on LPS‐primed primary human macrophages that were left untreated, or pre‐incubated with VHH ASC or mutVHH ASC (100 µg ml −1 ), CRID3 (50 µM) or VX‐765 (50 µM) for 30 min before being activated with (A) nigericin (10 µM), or (B) PFO (30 ng ml −1 ) for 2 h. Data is from the experiments displayed in Fig A and B. C Human IL‐1β (hIL‐1β) concentrations in cell‐free supernatants (left), and cell viability assay (right) of LPS‐primed primary human macrophages that were incubated with VHH ASC or mutVHH ASC (100 µg ml −1 ), CRID3 (50 µM) or VX‐765 (50 µM) for 30 min before being stimulated with 2.5 mM ATP. D hIL‐1β concentrations in cell‐free supernatants (top), and cell viability assay (bottom) of PMA‐differentiated THP‐1 cells treated with VHH ASC or mutVHH ASC (100 µg ml −1 ), CRID3 (10 µM) or VX‐765 (50 µM) for 30 min before 4.5 h stimulation with 250 µg ml −1 MSU crystals. E hIL‐1β concentrations in cell‐free supernatants (top), and cell viability assay (bottom) of LPS‐primed primary human macrophages that were incubated with VHH ASC or mutVHH ASC (100 µg ml −1 ), CRID3 (50 µM) or VX‐765 (50 µM) for 30 min before being stimulated with 0.1 µg ml −1 /0.5 µg ml −1 mixture of LFn‐BsaK and PA for 2 h. F hIL‐1β concentrations in cell‐free supernatants (top), and cell viability assay (bottom) of Pam3CysK4‐primed (1 µg ml −1 ) primary human CD14 + monocytes that were incubated with VHH ASC or mutVHH ASC (100 µg ml −1 ), or VX‐765 (50 µM) for 30 min before being stimulated with 1 µg ml −1 TcdA. G hIL‐1β concentrations in cell‐free supernatants (top), and cell viability assay (bottom) of keratinocyte cells (N‐TERT) that were treated with VHH ASC or mutVHH ASC (100 µg ml −1 ), or VX‐765 (50 µM), then directly stimulated with 30 µM Val‐boroPro (VbP) for 22 h. H hIL‐1β concentrations in cell‐free supernatants (top), and cell viability assay (bottom) of PMA‐differentiated THP‐1 cells treated with IFNγ (500 U ml −1 ) for 16 h and that were incubated with VHH ASC or mutVHH ASC (100 µg ml −1 ), CRID3 (10 µM) or VX‐765 (50 µM) for 30 min before 2 h stimulation with 1 µg ml −1 poly(dA:dT) in complex with Lipofectamine 2000. Data information: Data is representative of either two independent experiments, each run with one to two donors (A–C, E, F, 3 or 4 donors in total) or at least 3 independent experiments (D, G, H). Each symbol represents one donor or independent experiment. ns P > 0.05; * P < 0.05; ** P < 0.005; *** P < 0.0002; **** P < 0.0001, One‐way ANOVA, multiple comparison (Tukey test). Data is displayed as floating bars with the max/min values and mean (thicker band).

Techniques Used: CtB Assay, Incubation, Viability Assay, Comparison

A Specificity of different single‐domain antibodies (VHHs) probed by ELISA. Recombinant murine ASC as an eGFP fusion or eGFP alone (GFP‐LPETG) as control, were coated onto ELISA plates at 1 µg ml −1 /well. Wells were incubated with HA‐tagged VHHs (100 nM), anti‐HA‐tag mouse mAb coupled to HRP, and the HRP substrate TMB. Binding was quantified by measuring the absorbance at 450 nm. B, C Lysates of HEK 293T cells transiently expressing HA‐tagged VHH mASC or VHH ASC and the indicated bait proteins fused to Renilla luciferase were used to immunoprecipitate VHHs with immobilized anti‐HA antibody. Renilla luciferase activity of the co‐immunoprecipitated proteins was measured and normalized to the input luciferase. Data represents mean values ± SEM from three independent experiments. D, E Cell viability (CTB) assay on LPS‐primed (200 ng ml −1 ) primary mouse BMDMs that were left untreated, or pre‐incubated with VHH ASC or VHH mASC (100 µg ml −1 ), CRID3 (50 µM) or VX‐765 (50 µM) for 30 min before being activated with (D) nigericin (10 µM), or (E) PFO (250 ng ml −1 ) for 2 h. Data is from the experiments displayed in Fig C and D. Data is displayed as floating bars with the max/min values and mean (thicker band). F Mouse IL‐1β (mIL‐1β) concentrations in cell‐free supernatants (left), and cell viability assay (right) of LPS‐primed mouse BMDMs incubated with VHHs, CRID3 or VX–765, before stimulation with 0.1 µg ml −1 /0.5 µg ml −1 mixture of LFn‐BsaK and PA for 2 h. Each symbol represents one mouse. Data is displayed as floating bars with the max/min values and mean (thicker band). Data information: ns P > 0.05; *** P < 0.001; **** P < 0.0001, Two‐way (A, C) or One‐way (B, D–F) ANOVA, multiple comparison (Tukey test).

Figure Legend Snippet: A Specificity of different single‐domain antibodies (VHHs) probed by ELISA. Recombinant murine ASC as an eGFP fusion or eGFP alone (GFP‐LPETG) as control, were coated onto ELISA plates at 1 µg ml −1 /well. Wells were incubated with HA‐tagged VHHs (100 nM), anti‐HA‐tag mouse mAb coupled to HRP, and the HRP substrate TMB. Binding was quantified by measuring the absorbance at 450 nm. B, C Lysates of HEK 293T cells transiently expressing HA‐tagged VHH mASC or VHH ASC and the indicated bait proteins fused to Renilla luciferase were used to immunoprecipitate VHHs with immobilized anti‐HA antibody. Renilla luciferase activity of the co‐immunoprecipitated proteins was measured and normalized to the input luciferase. Data represents mean values ± SEM from three independent experiments. D, E Cell viability (CTB) assay on LPS‐primed (200 ng ml −1 ) primary mouse BMDMs that were left untreated, or pre‐incubated with VHH ASC or VHH mASC (100 µg ml −1 ), CRID3 (50 µM) or VX‐765 (50 µM) for 30 min before being activated with (D) nigericin (10 µM), or (E) PFO (250 ng ml −1 ) for 2 h. Data is from the experiments displayed in Fig C and D. Data is displayed as floating bars with the max/min values and mean (thicker band). F Mouse IL‐1β (mIL‐1β) concentrations in cell‐free supernatants (left), and cell viability assay (right) of LPS‐primed mouse BMDMs incubated with VHHs, CRID3 or VX–765, before stimulation with 0.1 µg ml −1 /0.5 µg ml −1 mixture of LFn‐BsaK and PA for 2 h. Each symbol represents one mouse. Data is displayed as floating bars with the max/min values and mean (thicker band). Data information: ns P > 0.05; *** P < 0.001; **** P < 0.0001, Two‐way (A, C) or One‐way (B, D–F) ANOVA, multiple comparison (Tukey test).

Techniques Used: Enzyme-linked Immunosorbent Assay, Recombinant, Control, Incubation, Binding Assay, Expressing, Luciferase, Activity Assay, Immunoprecipitation, CtB Assay, Viability Assay, Comparison

THP‐1 cells expressing a Dox‐inducible CRISPR‐Cas9 cassette targeting GSDMD were left untreated (–), or treated with 1 µg ml −1 Dox for one or two cycles of 72 h (1×, or 2× respectively). Immunoblot analysis of GSDMD expression following the indicated course of Dox treatment and PMA‐differentiation, as indicated. Data is from one representative of two independent experiments. IL‐1β concentration or percentage of LDH released into cell‐free supernatants of PMA‐differentiated THP‐1 cells that were treated with VHH ASC (200 µg ml −1 ) or CRID3 (25 µM) for 30 min prior to stimulation with nigericin (10 µM, left panels) or PFO (30 ng ml −1 , right panels) for 2 h. Data is average of experimental duplicates from three independent experiments, each represented by a different symbol. ns P > 0.05; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001, Two‐way ANOVA, multiple comparison (Tukey test). Data is displayed as floating bars with the max/min values and mean (thicker band). Live confocal imaging of PMA‐differentiated and nigericin‐treated (10 µM) THP‐1 cells expressing human ASC‐GFP (green) in the presence of AlexaFluor647‐labeled VHH ASC (VHH ASC ‐AF647, 10 µg ml −1 , cyan) in the medium. Cells were either left untreated (–) or incubated with VX‐765 (50 µM) for 1 h prior to nigericin stimulation. Nuclei were stained with Hoechst 34580 (magenta). Scale bar: 50 µm. Data is from one representative out of two independent experiments. Source data are available online for this figure.

Figure Legend Snippet: THP‐1 cells expressing a Dox‐inducible CRISPR‐Cas9 cassette targeting GSDMD were left untreated (–), or treated with 1 µg ml −1 Dox for one or two cycles of 72 h (1×, or 2× respectively). Immunoblot analysis of GSDMD expression following the indicated course of Dox treatment and PMA‐differentiation, as indicated. Data is from one representative of two independent experiments. IL‐1β concentration or percentage of LDH released into cell‐free supernatants of PMA‐differentiated THP‐1 cells that were treated with VHH ASC (200 µg ml −1 ) or CRID3 (25 µM) for 30 min prior to stimulation with nigericin (10 µM, left panels) or PFO (30 ng ml −1 , right panels) for 2 h. Data is average of experimental duplicates from three independent experiments, each represented by a different symbol. ns P > 0.05; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001, Two‐way ANOVA, multiple comparison (Tukey test). Data is displayed as floating bars with the max/min values and mean (thicker band). Live confocal imaging of PMA‐differentiated and nigericin‐treated (10 µM) THP‐1 cells expressing human ASC‐GFP (green) in the presence of AlexaFluor647‐labeled VHH ASC (VHH ASC ‐AF647, 10 µg ml −1 , cyan) in the medium. Cells were either left untreated (–) or incubated with VX‐765 (50 µM) for 1 h prior to nigericin stimulation. Nuclei were stained with Hoechst 34580 (magenta). Scale bar: 50 µm. Data is from one representative out of two independent experiments. Source data are available online for this figure.

Techniques Used: Expressing, CRISPR, Western Blot, Concentration Assay, Comparison, Imaging, Labeling, Incubation, Staining

Protease Inhibitor:

Article Title: A ubiquitin-independent proteasome pathway controls activation of the CARD8 inflammasome
Article Snippet: Reagents for immunoprecipitation experiments include anti-FLAG M2 agarose resin (Millipore, A2220) and Pierce anti-HA agarose resin (Thermo Scientific, 26181). .. Other reagents used include Val-boroPro (VbP; Tocris 3719), bortezomib (BORT; Millipore-Sigma, 504314), MG-132 (Sigma-Aldrich, 474787), bestatin methyl ester (MeBs; Sigma, 200485), MLN4924 (Cayman, 15217), dTAG13 (Tocris, 6605), VX-765 (Cayman, 28825), doxycycline hyclate (Cayman, 14422), FuGENE HD (Promega, E2311), and HALT protease inhibitor cocktail (Thermo Scientific, 78430). .. Human erythrocyte-derived, purified 20S proteasomes used are from South Bay Bio (SBB-PP0005), Aviva Systems Biology (OPED00120), Enzo Life Sciences (BML-PW8720-0050), and R&D Systems (E-360).

Article Title: A ubiquitin-independent proteasome pathway controls the CARD8 inflammasome
Article Snippet: Reagents for immunoprecipitation experiments include anti-FLAG M2 agarose resin (Millipore, A2220), Pierce anti-HA agarose resin (Thermo Scientific, 26181). .. Other reagents used include Val-boroPro (VbP; Tocris 3719), bortezomib (BORT; Millipore-Sigma, 504314), MG-132 (Sigma-Aldrich, 474787), bestatin methyl ester (MeBs; Sigma, 200485), MLN4924 (Cayman, 15217), dTAG13 (Tocris, 6605), VX-765 (Cayman, 28825), Doxycycline hyclate (Cayman, 14422), FuGENE HD (Promega, E2311), HALT protease inhibitor cocktail (Thermo Scientific, 78430). .. Human erythrocyte-derived, purified 20S proteasomes used are from South Bay Bio (SBB-PP0005), Aviva Systems Biology (OPED00120), Enzo Life Sciences (BML-PW8720-0050), and R&D Systems (E-360).

Incubation:

Article Title: Coptisine from Coptis chinensis blocks NLRP3 inflammasome activation by inhibiting caspase-1.
Article Snippet: Accepted Manuscript Title: Coptisine from Coptis chinensis blocks NLRP3 inflammasome activation by inhibiting caspase-1 Authors: Jiasi Wu, Yu Luo, Qing Jiang, Sheng Li, Wenge Huang, Li Xiang, Deming Liu, Yingfan Hu, Ping Wang, Xiaoxia Lu, Guolin Zhang, Fei Wang, Xianli Meng PII: S1043-6618(19)30323-8 DOI: https://doi.org/10.1016/j.phrs.2019.104348 Article Number: 104348 Reference: YPHRS 104348 To appear in: Pharmacological Research Received date: 25 February 2019 Revised date: 26 June 2019 Accepted date: 10 July 2019 Please cite this article as: Wu J, Luo Y, Jiang Q, Li S, Huang W, Xiang L, Liu D, Hu Y, Wang P, Lu X, Zhang G, Wang F, Meng X, Coptisine from Coptis chinensis blocks NLRP3 inflammasome activation by inhibiting caspase-1, Pharmacological Research (2019), https://doi.org/10.1016/j.phrs.2019.104348 This is a PDF file of an unedited manuscript that has been accepted for publication.. As a service to our customers we are providing this early version of the manuscript.. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form.

Enzyme-linked Immunosorbent Assay:

other:

Article Title: The NLRP3 inflammasome is essential for IL-18 production in a murine model of macrophage activation syndrome
Article Snippet: The following treatments were given (I.P. unless stated): MCC950 (50 mg/kg in PBS, ( )) (CP-456773 sodium salt, Sigma Aldrich) at the same time as CpG; vehicle controls received 10 μL/g PBS; VX765 (Belnacasan) (100mg/kg in 5% (v/v) DMSO in PBS, ( )) (273404-37-8, Tocris Bioscience) injected daily; vehicle controls received 10 μL/g 5% (v/v) DMSO in PBS; recombinant IL-1Ra (100mg/kg in placebo (638 mM polysorbate 80, 5.2 mM sodium citrate, 112 mM sodium chloride, 45.4 μM disodium EDTA, dH2O, pH 6.5)) (Anakinra, Sobi) administered twice daily ( ); high dose IL-1Ra recommended for clinical treatment of CSS ( , ) by sub-cutaneous (S.C) injection; vehicle controls received 10 μL/g placebo.

Article Title: HIV-1 infection causes depletion of monocytic cells through a non-canonical cell death pathway
Article Snippet: The drugs/chemicals used in this study are listed, along with the companies they were obtained from in parentheses: Nevirapine, and tenofovir (Merck), raltegravir (Santa Cruz), Ruxolitinib, AZD7648, necrosulfonamide (Selleckchem), Z-VAD-FMK, bafilomycin A1 (Invivogen), VX-765 (Tocris), MG132, chloroquine, digoxin (Sigma).

Image Search Results

Pre‐incubation with Pep19‐2.5 inhibits nigericin‐ and MSU crystal‐induced IL‐1β secretion. (A) Primary monocytes were primed with 1 µg/mL Pam 3 CSK 4 for 3 h and then stimulated with 10 µM nigericin or 200 µg/mL MSU crystals for 3 h. Pep19‐2.5 was added 30 min before priming at increasing concentrations ranging from 0.4 to 18 µM. Supernatants were analyzed for IL‐1β by ELISA. Stimulus‐induced IL‐1β secretion was set to 100%. Mean ± SEM ( n = 4 biologically independent experiments for nigericin, n = 2 biologically independent experiments for MSU crystals). One‐sample t ‐test against 100%. (B) hMDMs were primed with 1 µg/mL Pam 3 CSK 4 for 4 h and then stimulated with 5 µM nigericin for 2 h. Pep19‐2.5 was added at the indicated concentrations 30 min before priming. Supernatants were analyzed for IL‐1β by ELISA. Stimuli‐induced IL‐1β secretion was set to 100%. Mean + SEM ( n = 4 biologically independent experiments). One‐sample t ‐test against 100%. (C) THP‐1 macrophages were primed and stimulated as described in (A). For inhibition experiments, 18 µM Pep19‐2.5 was added 30 min before priming. Supernatants were analyzed for IL‐1β by ELISA. Stimulus‐induced IL‐1β secretion was set to 100%. Mean + SEM ( n = 4 biologically independent experiments for nigericin, n = 3 biologically independent experiments for MSU crystals). One‐sample t ‐test against 100%. (D and E) THP‐1 macrophages were primed and treated with NLRP1 (D) or AIM2 (E) activators. Supernatants were analyzed for IL‐1β by ELISA. Stimulus‐induced IL‐1β secretion was set to 100%. Mean + SEM ( n = 3 biologically independent experiments). One‐sample t ‐test against 100%. (F–H) Protein expression of pro‐IL‐1β (F), full‐length (G), and cleaved GSDMD (H) were analyzed by Western blot. Basal, Pam 3 CSK 4 ‐ or nigericin‐induced expression was set to 100%. Mean + SEM ( n = 3 biologically independent experiments). One‐sample t ‐test against 100%. (I) THP‐1 macrophages were primed and stimulated after peptide incubation as described in (A). After 1 h of stimulation with nigericin, the medium was removed, and Z‐WEHD buffer was added according to the manufacturer´s protocol. Ac‐YVAD‐CHO control was subtracted, and stimulus‐induced caspase‐1 activity was normalized to 100%. Mean ± SEM ( n = 3 biologically independent experiments). One‐sample t ‐test against 100%. (J) hMDMs from healthy donors were seeded in µ‐Slides VI and incubated at 37°C in a humidified atmosphere of 5% CO 2 for 1 h. Macrophages were primed with 1 µg/mL Pam 3 CSK 4 for 3 h and stimulated with 10 µM nigericin for 1 h in the presence of fluorophore‐conjugated Rh‐Pep19‐2.5. Active caspase‐1 was stained with the caspase‐1 pseudosubstrate FLICA660‐YVAD‐FMK. Cells were washed and fixed, and nuclei were stained with Hoechst 34580. Confocal microscopy images are representative of n = 2 biologically independent experiments. Scale bar is 10 µm (upper panel) and 2 µm (zoomed in lower panel). (K) Solutions containing 0.5 µM Atto488‐conjugated Pep19‐2.5 were prepared in the presence of increasing concentrations of recombinant human caspase‐1 and incubated overnight at 4°C under gentle agitation. Microfluidic diffusional sizing (MDS) measurements were performed at room temperature to determine the hydrodynamic radius ( R h ) of fluorescently labeled particles. Dots represent the mean ± SD of replicates ( n ≥ 3) of free peptide and peptide–protein complexes, and the black line represents the best nonlinear fit according to Equation . (L) Human recombinant caspase‐1 was incubated with the caspase‐1‐specific substrate Ac‐YVAD‐AMC at the indicated concentrations of Pep19‐2.5 or 40 µM of the caspase‐1 inhibitor VX765. Enzyme activity in relative fluorescence units (RFU) was measured every 2 min at 37°C. Representative traces are means ± SEM ( n = 2 biologically independent experiments).

Journal: Advanced Science

Article Title: Membrane‐Active Peptide Protects Against Inflammation by Targeting NLRP3 Activation at the Trans‐Golgi Network

doi: 10.1002/advs.76587

Figure Lengend Snippet: Pre‐incubation with Pep19‐2.5 inhibits nigericin‐ and MSU crystal‐induced IL‐1β secretion. (A) Primary monocytes were primed with 1 µg/mL Pam 3 CSK 4 for 3 h and then stimulated with 10 µM nigericin or 200 µg/mL MSU crystals for 3 h. Pep19‐2.5 was added 30 min before priming at increasing concentrations ranging from 0.4 to 18 µM. Supernatants were analyzed for IL‐1β by ELISA. Stimulus‐induced IL‐1β secretion was set to 100%. Mean ± SEM ( n = 4 biologically independent experiments for nigericin, n = 2 biologically independent experiments for MSU crystals). One‐sample t ‐test against 100%. (B) hMDMs were primed with 1 µg/mL Pam 3 CSK 4 for 4 h and then stimulated with 5 µM nigericin for 2 h. Pep19‐2.5 was added at the indicated concentrations 30 min before priming. Supernatants were analyzed for IL‐1β by ELISA. Stimuli‐induced IL‐1β secretion was set to 100%. Mean + SEM ( n = 4 biologically independent experiments). One‐sample t ‐test against 100%. (C) THP‐1 macrophages were primed and stimulated as described in (A). For inhibition experiments, 18 µM Pep19‐2.5 was added 30 min before priming. Supernatants were analyzed for IL‐1β by ELISA. Stimulus‐induced IL‐1β secretion was set to 100%. Mean + SEM ( n = 4 biologically independent experiments for nigericin, n = 3 biologically independent experiments for MSU crystals). One‐sample t ‐test against 100%. (D and E) THP‐1 macrophages were primed and treated with NLRP1 (D) or AIM2 (E) activators. Supernatants were analyzed for IL‐1β by ELISA. Stimulus‐induced IL‐1β secretion was set to 100%. Mean + SEM ( n = 3 biologically independent experiments). One‐sample t ‐test against 100%. (F–H) Protein expression of pro‐IL‐1β (F), full‐length (G), and cleaved GSDMD (H) were analyzed by Western blot. Basal, Pam 3 CSK 4 ‐ or nigericin‐induced expression was set to 100%. Mean + SEM ( n = 3 biologically independent experiments). One‐sample t ‐test against 100%. (I) THP‐1 macrophages were primed and stimulated after peptide incubation as described in (A). After 1 h of stimulation with nigericin, the medium was removed, and Z‐WEHD buffer was added according to the manufacturer´s protocol. Ac‐YVAD‐CHO control was subtracted, and stimulus‐induced caspase‐1 activity was normalized to 100%. Mean ± SEM ( n = 3 biologically independent experiments). One‐sample t ‐test against 100%. (J) hMDMs from healthy donors were seeded in µ‐Slides VI and incubated at 37°C in a humidified atmosphere of 5% CO 2 for 1 h. Macrophages were primed with 1 µg/mL Pam 3 CSK 4 for 3 h and stimulated with 10 µM nigericin for 1 h in the presence of fluorophore‐conjugated Rh‐Pep19‐2.5. Active caspase‐1 was stained with the caspase‐1 pseudosubstrate FLICA660‐YVAD‐FMK. Cells were washed and fixed, and nuclei were stained with Hoechst 34580. Confocal microscopy images are representative of n = 2 biologically independent experiments. Scale bar is 10 µm (upper panel) and 2 µm (zoomed in lower panel). (K) Solutions containing 0.5 µM Atto488‐conjugated Pep19‐2.5 were prepared in the presence of increasing concentrations of recombinant human caspase‐1 and incubated overnight at 4°C under gentle agitation. Microfluidic diffusional sizing (MDS) measurements were performed at room temperature to determine the hydrodynamic radius ( R h ) of fluorescently labeled particles. Dots represent the mean ± SD of replicates ( n ≥ 3) of free peptide and peptide–protein complexes, and the black line represents the best nonlinear fit according to Equation . (L) Human recombinant caspase‐1 was incubated with the caspase‐1‐specific substrate Ac‐YVAD‐AMC at the indicated concentrations of Pep19‐2.5 or 40 µM of the caspase‐1 inhibitor VX765. Enzyme activity in relative fluorescence units (RFU) was measured every 2 min at 37°C. Representative traces are means ± SEM ( n = 2 biologically independent experiments).

Article Snippet: For this purpose, Pep19‐2.5 or the specific caspase‐1 inhibitor VX765 (HY‐13205, MedChemExpress, NJ, USA) was added in triplicates to the assay buffer (pH = 7.4) consisting of 50 mM HEPES, 100 mM NaCl, 0.1% CHAPS (w/v), 1 mM EDTA, 10% glycerol (v/v), and 10 mM DTT.

Techniques: Incubation, Enzyme-linked Immunosorbent Assay, Inhibition, Expressing, Western Blot, Control, Activity Assay, Staining, Confocal Microscopy, Recombinant, Gentle, Labeling, Fluorescence

Pep19‐2.5 inhibits NLRP3‐induced ASC assembly but does not bind to NLRP3. (A) Representative Western blot of cross‐linked cytosolic pellet from primed THP‐1 macrophages that were pre‐incubated with increasing concentrations of Pep19‐2.5 and stimulated with 10 µM nigericin for 1 h ( n = 3 biologically independent experiments). (B) HEK293 ASC‐BFP cells transduced with NLRP3 were pre‐incubated with increasing concentrations of Pep19‐2.5 or MCC950 and stimulated with nigericin. ASC speck formation was determined by flow cytometry. Dot plots show mean ± SEM ( n = 3 biologically independent experiments). One‐way ANOVA followed by Dunnet's post‐test. (C) THP‐1 C1C‐EGFP macrophages were incubated with the indicated concentrations of Pep19‐2.5 for 30 min prior to priming. After priming with Pam 3 CSK 4 , 40 µM VX765 was added to limit cell death, and 10 µM nigericin was added to stimulate NLRP3 assembly for 1 h. Staining was performed using an anti‐ASC antibody and Hoechst 34580. Confocal microscopy images are representative of n = 2 biologically independent experiments, with three individual images taken for each condition. Scale bar = 20 µm. Full images are shown Figure . (D) Nuclei, ASC, and C1C specks in the images shown in (C) were quantified separately in the corresponding fluorescence channels. For each condition, three independent microscopy images were analyzed, with each data point representing a single image. Speck formation was quantified manually, and results are presented as mean + SEM. One‐way ANOVA followed by Šídák's multiple comparison post hoc analysis. (E) Melting temperature of NLRP3 NACHT alone, with the inhibitor MCC950 or Pep19‐2.5, as determined by nanoDSF ( n = 3 independent experiments). No shift upon addition of Pep19‐2.5 is observed, indicating no direct interaction of Pep19‐2.5 with the NACHT domain of NLRP3. (F) The thermal stability of the decameric NLRP3 (fl., wt) protein with and without Pep19‐2.5 was determined by measuring the hydrodynamic radius by DLS ( n = 3 independent experiments).

Journal: Advanced Science

Article Title: Membrane‐Active Peptide Protects Against Inflammation by Targeting NLRP3 Activation at the Trans‐Golgi Network

doi: 10.1002/advs.76587

Figure Lengend Snippet: Pep19‐2.5 inhibits NLRP3‐induced ASC assembly but does not bind to NLRP3. (A) Representative Western blot of cross‐linked cytosolic pellet from primed THP‐1 macrophages that were pre‐incubated with increasing concentrations of Pep19‐2.5 and stimulated with 10 µM nigericin for 1 h ( n = 3 biologically independent experiments). (B) HEK293 ASC‐BFP cells transduced with NLRP3 were pre‐incubated with increasing concentrations of Pep19‐2.5 or MCC950 and stimulated with nigericin. ASC speck formation was determined by flow cytometry. Dot plots show mean ± SEM ( n = 3 biologically independent experiments). One‐way ANOVA followed by Dunnet's post‐test. (C) THP‐1 C1C‐EGFP macrophages were incubated with the indicated concentrations of Pep19‐2.5 for 30 min prior to priming. After priming with Pam 3 CSK 4 , 40 µM VX765 was added to limit cell death, and 10 µM nigericin was added to stimulate NLRP3 assembly for 1 h. Staining was performed using an anti‐ASC antibody and Hoechst 34580. Confocal microscopy images are representative of n = 2 biologically independent experiments, with three individual images taken for each condition. Scale bar = 20 µm. Full images are shown Figure . (D) Nuclei, ASC, and C1C specks in the images shown in (C) were quantified separately in the corresponding fluorescence channels. For each condition, three independent microscopy images were analyzed, with each data point representing a single image. Speck formation was quantified manually, and results are presented as mean + SEM. One‐way ANOVA followed by Šídák's multiple comparison post hoc analysis. (E) Melting temperature of NLRP3 NACHT alone, with the inhibitor MCC950 or Pep19‐2.5, as determined by nanoDSF ( n = 3 independent experiments). No shift upon addition of Pep19‐2.5 is observed, indicating no direct interaction of Pep19‐2.5 with the NACHT domain of NLRP3. (F) The thermal stability of the decameric NLRP3 (fl., wt) protein with and without Pep19‐2.5 was determined by measuring the hydrodynamic radius by DLS ( n = 3 independent experiments).

Techniques: Western Blot, Incubation, Transduction, Flow Cytometry, Staining, Confocal Microscopy, Fluorescence, Microscopy, Comparison, Nano Differential Scanning Fluorimetry

Pep19‐2.5 reduces pro‐inflammatory IL‐1β response to house dust mite extract in vitro and in vivo. (A–E) Human macrophages from heathy donors were seeded in serum‐free Opti‐MEM medium and stimulated without priming with the indicated doses of house dust mite (HDM) extract for 24 h at 37°C (A) alone, in the presence of (B) the NLRP3 inhibitor MCC950 (10 µM), (C) the TLR4 antagonist eritoran (1 µg/mL), or (D–F) Pep19‐2.5. IL‐1β release was determined from cell‐free supernatants by ELISA. Data shown in (A) mean + SD of duplicate analyses and are representative for n = 7 independent donors. Data in (B–E) were normalized to 100% for HDM in the absence of inhibitors and are shown as mean + SEM of (B, C, E, F): n = 5 and (D): n = 3 independent experiments with cells from different healthy donors. Statistical significance against HDM alone was analyzed by a two‐sided t ‐test. (G) Treatment protocol for HDM‐induced allergic asthma mouse model. (H) Airway resistance in response to methacholine inhalation, (I) inflammatory cell infiltrate volume in lung tissue per epithelial basal membrane (b.m.) area, and (J) numbers of eosinophils on day 14 in bronchoalveolar lavage (BAL) fluid of healthy (PBS), asthmatic (HDM), or Pep19‐2.5‐treated mice (HDM + peptide), n = 10 mice per group. (K–O) Cytokine levels in BAL fluid were assessed by MSD U‐Plex assays. (P) Caspase‐1 mRNA expression in BAL cells and (Q) caspase‐1 protein determined by Western blot. (R) Final body weight on day 14 of the treatment protocol. Results are presented as mean values ± SEM. Statistical significance was assessed using ordinary one‐way ANOVA and Tukey´s multiple comparison post hoc analyses.

Journal: Advanced Science

Article Title: Membrane‐Active Peptide Protects Against Inflammation by Targeting NLRP3 Activation at the Trans‐Golgi Network

doi: 10.1002/advs.76587

Figure Lengend Snippet: Pep19‐2.5 reduces pro‐inflammatory IL‐1β response to house dust mite extract in vitro and in vivo. (A–E) Human macrophages from heathy donors were seeded in serum‐free Opti‐MEM medium and stimulated without priming with the indicated doses of house dust mite (HDM) extract for 24 h at 37°C (A) alone, in the presence of (B) the NLRP3 inhibitor MCC950 (10 µM), (C) the TLR4 antagonist eritoran (1 µg/mL), or (D–F) Pep19‐2.5. IL‐1β release was determined from cell‐free supernatants by ELISA. Data shown in (A) mean + SD of duplicate analyses and are representative for n = 7 independent donors. Data in (B–E) were normalized to 100% for HDM in the absence of inhibitors and are shown as mean + SEM of (B, C, E, F): n = 5 and (D): n = 3 independent experiments with cells from different healthy donors. Statistical significance against HDM alone was analyzed by a two‐sided t ‐test. (G) Treatment protocol for HDM‐induced allergic asthma mouse model. (H) Airway resistance in response to methacholine inhalation, (I) inflammatory cell infiltrate volume in lung tissue per epithelial basal membrane (b.m.) area, and (J) numbers of eosinophils on day 14 in bronchoalveolar lavage (BAL) fluid of healthy (PBS), asthmatic (HDM), or Pep19‐2.5‐treated mice (HDM + peptide), n = 10 mice per group. (K–O) Cytokine levels in BAL fluid were assessed by MSD U‐Plex assays. (P) Caspase‐1 mRNA expression in BAL cells and (Q) caspase‐1 protein determined by Western blot. (R) Final body weight on day 14 of the treatment protocol. Results are presented as mean values ± SEM. Statistical significance was assessed using ordinary one‐way ANOVA and Tukey´s multiple comparison post hoc analyses.

Techniques: In Vitro, In Vivo, Enzyme-linked Immunosorbent Assay, Membrane, Expressing, Western Blot, Comparison

IL-1β and TNF-α production in THP-1 cells and inflammasome-reconstructed 293T cells. (A) THP-1 cells were infected with the parental strain Nc1 or the NcGRA6-, NcGRA7-, or NcGRA14-deficient (KO) parasites at a multiplicity of infection (MOI) of 2.5 or treated with medium only (mock). At 20 h postinfection, the culture supernatants were collected for analysis. (B) 293T cells were transfected with inflammasome-reconstruction plasmids encoding NLRP3, ASC, procaspase-1, and pro-IL-1β, together with NcGRA7 cDNA or an empty vector. Untransfected 293T cells were used as a negative control (no plasmid). At 20 h posttransfection, the culture supernatants were collected for analysis. (C–H) THP-1 cells were pretreated with 10 μM MCC950 (an NLARP3 inhibitor), 100 μM VX765 (a CASP1 inhibitor), and 18 μM SN50 (an NF-κB inhibitor) for 2 hr and then infected with the Nc1 strain of N. caninum at a MOI of 2.5 or treated with medium only (mock). At 20 h postinfection, the culture supernatants were collected for analysis. Each value represents the mean ± SD of 4 replicates (technical replicates) in one representative experiment. Each experiment (biological replicate) was repeated two (G, H) , three (A, D, E, F) and four times (B, C) . Statistically significant differences according to one-way ANOVA or two-way ANOVA and a Tukey–Kramer post hoc analysis (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).

Journal: Frontiers in Immunology

Article Title: Mitochondrial damage and IL-1β production in monocytes caused by Neospora caninum infection are mediated by dense granule protein 7 and prohibitins

doi: 10.3389/fimmu.2025.1408992

Figure Lengend Snippet: IL-1β and TNF-α production in THP-1 cells and inflammasome-reconstructed 293T cells. (A) THP-1 cells were infected with the parental strain Nc1 or the NcGRA6-, NcGRA7-, or NcGRA14-deficient (KO) parasites at a multiplicity of infection (MOI) of 2.5 or treated with medium only (mock). At 20 h postinfection, the culture supernatants were collected for analysis. (B) 293T cells were transfected with inflammasome-reconstruction plasmids encoding NLRP3, ASC, procaspase-1, and pro-IL-1β, together with NcGRA7 cDNA or an empty vector. Untransfected 293T cells were used as a negative control (no plasmid). At 20 h posttransfection, the culture supernatants were collected for analysis. (C–H) THP-1 cells were pretreated with 10 μM MCC950 (an NLARP3 inhibitor), 100 μM VX765 (a CASP1 inhibitor), and 18 μM SN50 (an NF-κB inhibitor) for 2 hr and then infected with the Nc1 strain of N. caninum at a MOI of 2.5 or treated with medium only (mock). At 20 h postinfection, the culture supernatants were collected for analysis. Each value represents the mean ± SD of 4 replicates (technical replicates) in one representative experiment. Each experiment (biological replicate) was repeated two (G, H) , three (A, D, E, F) and four times (B, C) . Statistically significant differences according to one-way ANOVA or two-way ANOVA and a Tukey–Kramer post hoc analysis (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).

Article Snippet: MCC950 (an inhibitor of NLRP3 inflammasome activation by the inhibition of IL-1β release; Cayman Chemical), VX765 (an inhibitor of NLRP3 inflammasome activation by the inhibition of caspase 1 and IL-1β cleavage and release; LKT Laboratories), SN50 (an inhibitor of the nuclear transcription factor κB; Selleck, Houston, TX, USA), a carbonyl cyanide m-chlorophenyl hydrazone (CCCP, a uncoupling agent for oxidative phosphorylation that inhibits mitochondrial function; FUJFILM Wako Pure Chemical Corporation), rocaglamid A (Cayman, Ann Arbor, MI, USA) with biochemical research grades were used in this study.

Techniques: Infection, Transfection, Plasmid Preparation, Negative Control

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