Journal: Nature immunology

Article Title: E-selectin-mediated rapid NLRP3 inflammasome activation regulates S100A8/S100A9 release from neutrophils via transient gasdermin D pore formation.

doi: 10.1038/s41590-023-01656-1

Figure Lengend Snippet: Fig. 2 | E-selectin induces NLRP3 inflammasome activation in neutrophils. a, Schematic of the experimental design; PBMCs, peripheral blood mononuclear cells. b,c, E-selectin-induced cleavage of caspase 1 was assessed in isolated human neutrophils treated for 10 min with PBS/paquinimod/TAC242, PBS (control), E-selectin or a combination of E-selectin/paquinimod/TAC242 (b) and with PBS, E-selectin and E-selectin in the presence MCC950 (c). The amount of processed caspase 1 (casp-1 p20 and casp-1 p22) was determined in the supernatants, and the amount of procaspase 1 and GAPDH was determined in cell lysates (n = 4 independent experiments); Paq, paquinimod. d–f, Caspase 1 activity was determined in isolated human neutrophils stimulated with E-selectin or PBS (control) for 10 min loaded with FLICA dye and analyzed by confocal microscopy. Representative confocal images (d), quantification of mean FLICA+ area (e) and overall mean fluorescence intensity (MFI) of FLICA signal per cell

Article Snippet: In a second set of experiments, WT mice were pretreated with an intraperitoneal injection of MCC950 (10 mg per kg (body weight); Invivogen) or vehicle control 1 h before TNF injection.

Techniques: Activation Assay, Isolation, Control, Activity Assay, Confocal Microscopy, Fluorescence

Journal: Nature immunology

Article Title: E-selectin-mediated rapid NLRP3 inflammasome activation regulates S100A8/S100A9 release from neutrophils via transient gasdermin D pore formation.

doi: 10.1038/s41590-023-01656-1

Figure Lengend Snippet: Fig. 5 | E-selectin-induced GSDMD pore formation is transient. a, Schematic of the experimental design. b, Representative confocal images of PI uptake in isolated human neutrophils stimulated with PBS, E-selectin or E-selectin in the presence of MCC950. c,d, Time course analysis (c) and percentage of PI+ cells after 10 min of stimulation (d; 1,029 (PBS), 429 (E-selectin) and 315 (E-selectin/ MCC950) cells of n = 3 (control and E-selectin + MCC950) and 4 (E-selectin) independent experiments); Ft, fluorescence at the respective time point; F0, fluorescence at t0. e, Representative confocal images of PI uptake in bone marrow neutrophils from WT and Casp1−/−Casp11−/− mice treated with E-selectin. f,g, Time course (f) and percentage of PI+ WT and Casp1−/−Casp11−/− neutrophils after 10 min of stimulation (g; 190 (WT) and 351 (Casp1−/−Casp11−/−) cells of n = 3 mice per group). h–j, Cell death of human neutrophils was assessed via LDH release after stimulation with PBS or E-selectin for 10 min (h), 30 min (i) and

Article Snippet: In a second set of experiments, WT mice were pretreated with an intraperitoneal injection of MCC950 (10 mg per kg (body weight); Invivogen) or vehicle control 1 h before TNF injection.

Techniques: Isolation, Control, Fluorescence

Journal: Scientific Reports

Article Title: Remimazolam alleviates cerebral ischemia–reperfusion injury of rats by inhibiting NF-κB/NLRP3 inflammasome pyroptosis

doi: 10.1038/s41598-025-31205-9

Figure Lengend Snippet: Remimazolam reduces cortical neuronal NLRP3/ASC/Caspase-1 inflammasome-dependent pyroptosis by inhibiting NF-κB pathway activation. ( a ) The protein expressions of the NF-κB p65, NLRP3, and ASC were significantly reduced in the JSH-23 group, however, the protein expression of the NF-κB p65 was not significantly changed in the MCC950 group compared with the MCAO group. ( b ) Fluorescence intensity (green staining) of NF-κB p65, NLRP3, GSDMD, and Caspase-1 p20. Nuclei were stained with DAPI. Bar: 20 μm. ( c , d ) the proteins expressions of NF-κB p65, NLRP3, ASC, and caspase-1 p20 and the mRNAs of NF-κB p65, NLRP3, ASC, GSDMD, and caspase-1 p20 in vitro. ( e ) IL-1β secretion by ELISA assays both in vitro and in vivo studies. All tests were repeated independently three times. Data are presented as Mean ± SEM. ns: no significant; * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001: significant as compared to Sham group or control group; # p < 0.05, ## p < 0.01, ### p < 0.001, and #### p < 0.0001: significant as compared to MCAO group or OGD group.

Article Snippet: MCC950 (Selleck, S7809, USA), a select NLRP3 inhibition, was administered as the group MCC950 , .

Techniques: Activation Assay, Expressing, Fluorescence, Staining, In Vitro, Enzyme-linked Immunosorbent Assay, In Vivo, Control

Journal: BMC Complementary Medicine and Therapies

Article Title: Inhibitory effects of the flavonoids extracted from Pollen Typhae on palmitic acid-induced NLRP3 inflammasome activation in macrophages involving AMPK-mediated lipid metabolism

doi: 10.1186/s12906-025-05024-4

Figure Lengend Snippet: Pollen Typhae flavonoids attenuate PA-induced NLRP3 inflammasome activation in macrophages. ( A ) After pretreatment with or without LPS (1 ng/ml) for 3 h, RAW264.7 macrophages were stimulated with PA (0.125 ~ 0.5 mM) for 16 h, IL-1β and IL-18 in culture supernatants were analyzed by ELISA. ( B ) The macrophages were pretreated with LPS (1 ng/ml) for 3 h, followed by PA (0.5 mM) treatment for 16 h, the protein expression of NLRP3, Caspase-1, and Caspase-1 p10 was analyzed by western blotting. The uncropped blots were presented in Supplementary Fig. . ( C ) RAW264.7 macrophages were treated with PTF (0 ~ 0.5 mg/ml), TYP (0 ~ 100 µM) or I3ON (0 ~ 100 µM) for 16 and 20 h, the cell viability was detected by CCK-8 assay. * P < 0.05, ** P < 0.01 versus 0.0 mg/ml PTF (0.0 µM TYP or I3ON). ( D ) The macrophages were pretreated with LPS (1 ng/ml) or plus different concentrations of PTF (0.03125 ~ 0.5 mg/ml), TYP (10 ~ 100 µM), I3ON (10 ~ 100 µM) for 3 h before exposure to PA (0.5 mM) for 16 h, IL-1β and IL-18 in culture supernatants were checked by ELISA. ( E and F ) The macrophages were pretreated with LPS (1 ng/ml) or plus PTF (0.5 mg/ml), TYP (100 µM), AICAR (2 mM), MCC950 (10 µM) for 3 h followed by exposure to PA (0.5 mM) for 16 h, the protein (E) and gene expression (F) of NLRP3, Caspase-1 and IL-1β were checked by western blotting and Real-time PCR, respectively. The uncropped/unedited blots were presented in Supplementary Fig. . Each column shows the mean with SD ( n = 3–7). * P < 0.05, ** P < 0.01 versus LPS (1 ng/ml); # P < 0.05, ## P < 0.01 versus LPS (1 ng/ml) plus PA (0.5 mM)

Article Snippet: The macrophages were pre-exposed to LPS (1 ng/ml) or/and PTF (0.5 mg/ml), TYP (100 μM), AICAR (2 mM, Beyotime, Shanghai, China), MCC950 (10 μM, Cell Signaling Technology, Danvers, MA, USA), PF-05175157 (10 μM, GlpBio Technology, Montclair, CA, USA) for 3 h before treatment with PA (0.5 mM) for 16 h. The cells were harvested to obtain total protein.

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot, CCK-8 Assay, Gene Expression, Real-time Polymerase Chain Reaction

Journal: BMC Complementary Medicine and Therapies

Article Title: Inhibitory effects of the flavonoids extracted from Pollen Typhae on palmitic acid-induced NLRP3 inflammasome activation in macrophages involving AMPK-mediated lipid metabolism

doi: 10.1186/s12906-025-05024-4

Figure Lengend Snippet: Effects of PTF, AMPK agonist, ACC inhibitor and antioxidant on PA-induced Caspase-1 activity in macrophages. RAW264.7 macrophages were pretreated with LPS (1 ng/ml) or plus PTF (0.5 mg/ml), TYP (100 µM), PF-05175157 (10 µM), AICAR (2 mM), Compound C (10 µM), NAC (10 mM), MCC950 (10 µM) for 3 h before treatment with PA (0.5 mM) for 16 h, the Caspase-1 activity in culture supernatants ( A ) and the macrophages ( B ) was analyzed by bioluminescent method. Each column shows the mean with SD ( n = 3). * P < 0.05, ** P < 0.01 versus LPS (1 ng/ml); # P < 0.05, ## P < 0.01 versus LPS (1 ng/ml) plus PA (0.5 mM); § P < 0.05 versus LPS (1 ng/ml) plus PA (0.5 mM) and PTF (0.5 mg/ml)

Article Snippet: The macrophages were pre-exposed to LPS (1 ng/ml) or/and PTF (0.5 mg/ml), TYP (100 μM), AICAR (2 mM, Beyotime, Shanghai, China), MCC950 (10 μM, Cell Signaling Technology, Danvers, MA, USA), PF-05175157 (10 μM, GlpBio Technology, Montclair, CA, USA) for 3 h before treatment with PA (0.5 mM) for 16 h. The cells were harvested to obtain total protein.

Techniques: Activity Assay

Journal: eLife

Article Title: ENaC-mediated sodium influx exacerbates NLRP3-dependent inflammation in cystic fibrosis

doi: 10.7554/eLife.49248

Figure Lengend Snippet: Human bronchial epithelial cell (HBEC) lines (BEAS-2B (WT), IB3-1 (ΔF508/W1282X), CuFi1 (ΔF508/ ΔF508), CuFi4 (ΔF508/G551D) (n = 3 independent experiments) were unstimulated or stimulated with Lipopolysaccharide, from Escherichia coli K12 (LPS Ultrapure), which specifically targets TLR4 (10 ng/mL) for 4 hr before being stimulated for 4 hr with Flagellin (10 ng/mL with Lipofectamine 2000) for NLRC4 inflammasome, TcdB (10 ng/mL) for Pyrin inflammasome or poly(dA:dT) dsDNA (1 μg/mL with Lipofectamine 2000) for AIM2 inflammasome. ELISA assays were used to detect ( A ) IL-18. To monitor NLRP3 inflammasome activation, HBEC (n = 3 independent experiments) were pre-incubated with MCC950 (15 μM), OxPAPC (30 μg/mL) and YVAD (2 μg/mL) for 1 hr before a stimulation with LPS (10 ng/mL, 4 hr), and ATP (5 mM) for the final 30 min. ELISA assays were used to detect ( B ) IL-18 and ( D ) colourimetric assay used to detect caspase-1 activity in protein lysates for LPS/ATP and LPS/ATP/MCC950). ( D ) Necrosis and pyroptosis are represented as superimposed bar charts. Total necrosis was measured using LDH release assay. For pyroptotic cell death, each sample/condition was repeated in parallel with a caspase-1 inhibitor (YVAD (2 mg/mL, 1 hr)) pre-treatment. The total necrosis level was then taken away from the caspase-1 inhibited sample, or ‘caspase-1 independent’ necrosis, with the remaining LDH level termed ‘caspase-1 dependent necrosis’ or pyroptosis. Cells were then stimulated with LPS (10 ng/mL, 4 hr), and ATP (5 mM) for final 30 min. The assay was performed with HBEC lines (n = 3 independent experiments). (◦) Significance for Total Necrosis (●) Significance for Pyroptosis. A 2-way ANOVA with Tukey’s multiple comparison test was performed (p values * =< 0.05, ** =< 0.01, *** =< 0.001 and **** =< 0.0001).

Article Snippet: Cells were pre-treated with the following compounds where indicated prior to NLRP3 stimulation; MCC950 (15 nM, Cayman Chemical, Cambridge, UK) for 1 hr, YVAD (2 μg/ mL, Invivogen, San Diego, California) for 1 hr, OxPAPC (30 μg/ mL, Invivogen) for 1 hr, Amiloride (10 μM, 100 μM, Cayman Chemical, Cambridge, UK) for 1 hr, EIPA (10 μM, Cayman Chemical) for 1 hr, SPLUNC1-derived peptide, S18 (25 μM, gift from Spyryx Biosciences, Inc) for 4 hr or ouabain (100 nM, Cayman Chemical, Cambridge, UK) for 24 hr.

Techniques: Enzyme-linked Immunosorbent Assay, Activation Assay, Incubation, Activity Assay, Lactate Dehydrogenase Assay, Comparison

Journal: eLife

Article Title: ENaC-mediated sodium influx exacerbates NLRP3-dependent inflammation in cystic fibrosis

doi: 10.7554/eLife.49248

Figure Lengend Snippet: Primary monocytes from HC and CF (HC, n = 10; CF, n = 10) were unstimulated or stimulated with LPS which specifically targets TLR4 (10 ng/mL) for 4 hr before being stimulated for 4 hr with Flagellin (10 ng/mL with Lipofectamine 2000) for NLRC4 inflammasome, or TcdB (10 ng/mL) for Pyrin inflammasome or poly(dA:dT) dsDNA (1 μg/mL with Lipofectamine 2000) for AIM2 inflammasome. ELISA assays were used to detect ( A ) IL-18 and ( B ) IL-1β cytokine secretion in supernatants. To monitor NLRP3 inflammasome activation, primary monocytes from HC, CF, SAID and NCFB (HC, n = 10; CF, n = 10; SAID, n = 4; NCFB, n = 4) were pre-incubated with MCC950 (15 μM), OxPAPC (30 μg/mL) and YVAD (2 μg/mL) for 1 hr before a stimulation with LPS (10 ng/mL, 4 hr), and ATP (5 mM) for the final 30 min. ELISA assays were used to detect ( C ) IL-18 and ( D ) IL-1β cytokine secretion in supernatants and ( E ) a colourimetric assay was used to detect caspase-1 activity in protein lysates (HC, n = 10; CF, n = 10; SAID, n = 4; NCFB, n = 4). ( F ) Flow cytometry was used to detect ASC specks in supernatants of primary monocytes from HC, CF, SAID and NCFB (HC, n = 10; CF, n = 10; SAID, n = 6; NCFB, n = 4) for ±LPS/ATP and (HC, n = 5; CF, n = 5) for MCC950 with LPS/ATP. ( G ) Necrosis and pyroptosis are represented as superimposed bar charts. Total necrosis was measured using LDH release assay. For pyroptotic cell death, each sample/condition was repeated in parallel with a caspase-1 inhibitor (YVAD (2 mg/mL, 1 hr)) pre-treatment. The total necrosis level was taken away from the caspase-1 inhibited sample, or ‘caspase-1 independent’ necrosis, with the remaining LDH level termed ‘caspase-1 dependent necrosis’ or pyroptosis. Cells were then stimulated with LPS (10 ng/mL, 4 hr), and ATP (5 mM) for final 30 min. The assay was performed with primary monocytes from HC, CF, SAID and NCFB (HC, n = 10; CF, n = 10; SAID, n = 4; NCFB, n = 4). (◦) Significance for Total Necrosis (●) Significance for pyroptosis. A 2-way ANOVA statistical test was performed, with Tukey post-hoc correction (p values * =< 0.05, ** =< 0.01, *** =< 0.001 and **** =< 0.0001; error bars ± SEM). Inhibitor treatments in panels a-c were found to significantly reduce cytokine secretion and caspase-1 activity to **p =< 0.01 or less, for CF and SAID groups respectively. Significance values not displayed on the graph.

Article Snippet: Cells were pre-treated with the following compounds where indicated prior to NLRP3 stimulation; MCC950 (15 nM, Cayman Chemical, Cambridge, UK) for 1 hr, YVAD (2 μg/ mL, Invivogen, San Diego, California) for 1 hr, OxPAPC (30 μg/ mL, Invivogen) for 1 hr, Amiloride (10 μM, 100 μM, Cayman Chemical, Cambridge, UK) for 1 hr, EIPA (10 μM, Cayman Chemical) for 1 hr, SPLUNC1-derived peptide, S18 (25 μM, gift from Spyryx Biosciences, Inc) for 4 hr or ouabain (100 nM, Cayman Chemical, Cambridge, UK) for 24 hr.

Techniques: Enzyme-linked Immunosorbent Assay, Activation Assay, Incubation, Activity Assay, Flow Cytometry, Lactate Dehydrogenase Assay

Journal: eLife

Article Title: ENaC-mediated sodium influx exacerbates NLRP3-dependent inflammation in cystic fibrosis

doi: 10.7554/eLife.49248

Figure Lengend Snippet:

Article Snippet: Cells were pre-treated with the following compounds where indicated prior to NLRP3 stimulation; MCC950 (15 nM, Cayman Chemical, Cambridge, UK) for 1 hr, YVAD (2 μg/ mL, Invivogen, San Diego, California) for 1 hr, OxPAPC (30 μg/ mL, Invivogen) for 1 hr, Amiloride (10 μM, 100 μM, Cayman Chemical, Cambridge, UK) for 1 hr, EIPA (10 μM, Cayman Chemical) for 1 hr, SPLUNC1-derived peptide, S18 (25 μM, gift from Spyryx Biosciences, Inc) for 4 hr or ouabain (100 nM, Cayman Chemical, Cambridge, UK) for 24 hr.

Techniques: Isolation, Bicinchoninic Acid Protein Assay, Protease Inhibitor, Western Blot, Reverse Transcription, Recombinant, Plasmid Preparation, Software

Journal: Frontiers in Cellular Neuroscience

Article Title: Electroacupuncture Ameliorates Mechanical Allodynia of a Rat Model of CRPS-I via Suppressing NLRP3 Inflammasome Activation in Spinal Cord Dorsal Horn Neurons

doi: 10.3389/fncel.2022.826777

Figure Lengend Snippet: EA's anti-allodynic effect on CPIP rats is mimicked by pharmacological blocking NLRP3 inflammasome. (A) Experimental protocol. (B) 50% PWT following EA or MCC950 intervention (30 μg/rat/day, i.t.). (C) Normalized AUC deduced from (B) . (D) Immunostaining showing the effect of EA/MCC950 intervention on astrocyte (Upper panels, marked with GFAP) and microglia overactivation in SCDH. (E,F) Summary of the mean fluorescence intensity of GFAP (E) and OX42 (F) , normalized with the value of sham group. n = 5 rats/group. Scale bar indicates 100 μm. ** p < 0.01 # p < 0.05, ## p < 0.01. Two-way ANOVA with Tukey's post-hoc test was applied in panel B. One-way ANOVA with Tukey's post-hoc test was applied in (C,E,F) . NS, no significance.

Article Snippet: NLPR3 specific antagonist MCC950 and the activator nigericin were purchased from APExBIO Technology (USA).

Techniques: Blocking Assay, Immunostaining, Fluorescence