Journal: Veterinary Research

Article Title: Staphylococcus aureus mediates pyroptosis in bovine mammary epithelial cell via activation of NLRP3 inflammasome

doi: 10.1186/s13567-022-01027-y

Figure Lengend Snippet: NLRP3 inflammasome regulates ASC speck formation during S. aureus . Fluorescence microscopy images of MAC-T cells immunoassayed for ASC (red) with or without MCC950 after 4 h of S. aureus treatment.

Article Snippet: Caspase-1 inhibitor VX765 and NLRP3 inflammasome inhibitor MCC950 were obtained from Glpbio (USA) and used at final concentrations of 100 and 15 μM.

Techniques: Fluorescence, Microscopy

Journal: Veterinary Research

Article Title: Staphylococcus aureus mediates pyroptosis in bovine mammary epithelial cell via activation of NLRP3 inflammasome

doi: 10.1186/s13567-022-01027-y

Figure Lengend Snippet: NLRP3 inflammasome activation by S. aureus is essential for the generation of GSDMD-N and the release of IL-1β and IL-18. A Activated caspase-1 and B GSDMD-N or released C IL-1β and D IL-18 of MAC-T cells treated with or without NLRP3 inhibitor MCC950 or caspase-1 inhibitor VX765 for 1.5 h prior to treatment with S. aureus for 4 h. E Levels of IL-1β and F IL-18 released after S. aureus treatment of MAC-T cells for 4 h in the presence or absence of 25, 50, or 75 mM KCl.

Article Snippet: Caspase-1 inhibitor VX765 and NLRP3 inflammasome inhibitor MCC950 were obtained from Glpbio (USA) and used at final concentrations of 100 and 15 μM.

Techniques: Activation Assay

Journal: bioRxiv

Article Title: Heightened NLRP3 inflammasome activation is associated with aging and CMML diseases severity

doi: 10.1101/2022.01.27.477992

Figure Lengend Snippet: (A) Monocytes from younger and older healthy donors were stimulated with Pam3CSK4 (1 μg/mL) for different time points as indicated. Total cell lysates were analyzed for NF-κB activation by western blot with indicated antibodies. Blots are representative of two independent experiments. ( B) Monocytes from younger and older healthy donors were stimulated with Pam3CSK4 (2 μg/mL) or LPS (2 μg/mL) for 18 hours. Supernatants were analyzed for TNF by ELISA. Data is shown as mean ± SEM. Statistical analyses were performed using a two-tailed t -test. ( C-F ) Monocytes from younger and older healthy donors were pre-treated with or without dexamethasone (100 nM; NF-κB inhibitor) or MCC950 (2 μM; NLRP3 inhibitor) for 1 hour. After, cells were stimulated with LPS (2 μg/mL) or Pam3CSK4 (2 μg/mL) for 16 hours and with or without 5 μM of nigericin for 2 hours. Supernatants were analyzed for IL-1β (C, D) and TNF (E, F) by ELISA. Data is shown as mean ± SEM. Statistical analyses were performed using a two-tailed t -test. ( G and H ) Monocytes from younger and older healthy donors were pre-treated with or without Z-IETD FMK (4 μM; caspase-8 inhibitor) for 1 hour. Later cells were stimulated with LPS (2 μg/mL) or Pam3CSK4 (2 μg/mL) for 16 hours and stimulated for 2 hours with or without 5 μM of nigericin. Supernatants were analyzed for IL-1β (G) and TNF (H) by ELISA. Data is shown as mean ± SEM. Statistical analyses were performed using a two-tailed t -test. ( I ) Schematic figure showing that Pam3CSK4 directly activates NLRP3 inflammasome in monocytes from older but not from younger healthy donors. This activation is akin to LPS inducing the alternative NLRP3 inflammasome might depend on caspase 8.

Article Snippet: We purchased nigericin (N7143) and dexamethasone from Sigma-Aldrich, ultrapure LPS (LPS-EK), and Pam3Cysk4 (tlrl-pms) from Invivogen, Z-VAD-FMK (ALX-260-020) from Enzolifesciences, and MCC950 from Adipogen.

Techniques: Activation Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Heightened NLRP3 inflammasome activation is associated with aging and CMML diseases severity

doi: 10.1101/2022.01.27.477992

Figure Lengend Snippet: ( A-C ) Monocytes from older healthy donors (60 - 65 years of age, OHD) and CMML patients were stimulated with LPS (2 μg/mL) or Pam3CSK4 (2 μg/mL) for 16 hours and with or without 5 μM nigericin for 2 hours. Supernatants were analyzed for IL-1β (A) and TNF (B) by ELISA. Data is shown as mean ± SEM. Statistical analysis was performed using a two-tailed t -test. Cell lysates and supernatants were analyzed for pro- and cleaved-forms of caspase-1 and IL-1β by western blot (C). Blots are representative of three independent experiments. ( D and E ) Monocytes from older healthy donors and CMML patients were pre-treated with or without dexamethasone (100 nM; NF-κB inhibitor) or MCC950 (2 μM; NLRP3 inhibitor) or Z-IETD FMK (4 μM; caspase-8 inhibitor) for 1 hour. After, cells were stimulated with LPS (2 μg/mL) or Pam3CSK4 (2 μg/mL) for 16 hours and with or without 5 μM of nigericin for 2 hours. Supernatants were analyzed for IL-1β (D) and TNF (E) by ELISA. Data is shown as mean ± SEM. Statistical analysis was performed using a two-tailed t -test.

Article Snippet: We purchased nigericin (N7143) and dexamethasone from Sigma-Aldrich, ultrapure LPS (LPS-EK), and Pam3Cysk4 (tlrl-pms) from Invivogen, Z-VAD-FMK (ALX-260-020) from Enzolifesciences, and MCC950 from Adipogen.

Techniques: Enzyme-linked Immunosorbent Assay, Two Tailed Test, Western Blot

Journal: Frontiers in Stroke

Article Title: NLRP3 inflammasome inhibition protects against intracranial aneurysm rupture and alters the phenotype of infiltrating macrophages

doi: 10.3389/fstro.2023.1202137

Figure Lengend Snippet: MCC950 treatment reduces the aneurysm rupture rate and extends symptom-free survival. (A) Aneurysm formation rates, (B) Aneurysm rupture rate, * p < 0.05 by Fisher's exact test. (C) Cumulative symptom-free survival, and (D) Mean arterial pressure, p = n.s. at all-time points.

Article Snippet: MCC950 was obtained from Selleckchem (Catalog No.S7809, Houston, TX) and diluted in 0.9% NaCl in water.

Techniques:

Journal: Frontiers in Stroke

Article Title: NLRP3 inflammasome inhibition protects against intracranial aneurysm rupture and alters the phenotype of infiltrating macrophages

doi: 10.3389/fstro.2023.1202137

Figure Lengend Snippet: MCC950 treatment alters macrophage phenotype ratio in cerebral vasculature after aneurysm induction. (A) Representative images of M1-type (top) and M2-type (bottom) macrophages in saline-treated (left) and MCC950-treated (right) mice, scale bar = 50 μm. (B) Ratio of M1:M2 macrophage phenotypes in the vessel wall, * p < 0.05, n = 5 each. (C) Total number of F4/80 + cell per high-powered field, n = 5 each. All plots presented as median + 95% CI.

Article Snippet: MCC950 was obtained from Selleckchem (Catalog No.S7809, Houston, TX) and diluted in 0.9% NaCl in water.

Techniques: Saline

Journal: International journal of molecular sciences

Article Title: Inhibition of the NF-κB Signaling Pathway Alleviates Pyroptosis in Bladder Epithelial Cells and Neurogenic Bladder Fibrosis.

doi: 10.3390/ijms241311160

Figure Lengend Snippet: Figure 6. Bioinformatics analysis of the transcriptome sequencing results of cells. (A,B) Heatmap analysis of IRGs in cellular DEGs. (C–E) Expression levels of the pyroptosis proteins NLRP3 and ASC in SV-HUC-1 cells were detected by immunofluorescence staining (scale bar = 100 µm). (F) SEM showed TGF-β1-induced epithelial cell swelling, cell membrane pore formation, and py- roptotic body generation. Scale bar = 5 µm. (G) TEM showed cell membrane disruption, disruption of organelle structures such as mitochondria and the endoplasmic reticulum, and vacuolization. Scale bar = 2 µm and scale bar = 500 nm. (H) Protein expression levels of NLRP3, GSDMD, ASC, and IL1-β were detected by Western blotting in vitro. The data are presented as the mean ± SD; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The pyroptosis inhibitor MCC950 (10 μM, MCE, HY-12815A) was used to inhibit pyroptosis in SV-HUC-1 cells for 2 h before TGF-β1 stimulation [37,38].

Techniques: Sequencing, Expressing, Staining, Membrane, Disruption, Western Blot, In Vitro

Journal: Mediators of Inflammation

Article Title: Targeting NLRP3 Inflammasome Alleviates Synovitis by Reducing Pyroptosis in Rats with Experimental Temporomandibular Joint Osteoarthritis

doi: 10.1155/2022/2581151

Figure Lengend Snippet: Sequences of primers.

Article Snippet: The NLRP3 inhibitor MCC950 (MedChemExpress, Monmouth, NJ, USA) was diluted to 2 mg/ml with sterile saline and injected 10 mg/kg intraperitoneally.

Techniques:

Journal: Mediators of Inflammation

Article Title: Targeting NLRP3 Inflammasome Alleviates Synovitis by Reducing Pyroptosis in Rats with Experimental Temporomandibular Joint Osteoarthritis

doi: 10.1155/2022/2581151

Figure Lengend Snippet: Elevated expression of molecules in TMJ synovium. (a, b) Western blotting results of NLRP3, ASC, cleaved caspase-1, IL-1 β , and GSDMD in the sham and OA synovitis rats. (c) mRNA levels of NLRP3, ASC, caspase-1, IL-1 β , and GSDMD expressed in TMJ synovium between the sham and OA rats. (d) Immunohistochemistry of TMJ slices from the sham or OA rats using anti-NLRP3, anti-caspase-1, anti-GSDMD, and anti-IL-1 β antibodies, scale bar = 50 μ m. Positive cells are mainly around synovial membrane and vessels (black arrow). (e) Percentage of positive cells. Data in this figure are analyzed by independent samples t -test and presented as mean ± SD. ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001.

Article Snippet: The NLRP3 inhibitor MCC950 (MedChemExpress, Monmouth, NJ, USA) was diluted to 2 mg/ml with sterile saline and injected 10 mg/kg intraperitoneally.

Techniques: Expressing, Western Blot, Immunohistochemistry, Membrane

Journal: Mediators of Inflammation

Article Title: Targeting NLRP3 Inflammasome Alleviates Synovitis by Reducing Pyroptosis in Rats with Experimental Temporomandibular Joint Osteoarthritis

doi: 10.1155/2022/2581151

Figure Lengend Snippet: The biological effect of AYC and MCC950 on inflammasome- and pyroptosis-related molecules. (a, b) Western blotting results showing the protein levels of NLRP3, ASC, cleaved caspase-1, IL-1 β , and GSDMD among the sham, OA, and AYC groups. (a, c) Levels of the above proteins among the sham, OA, and MCC950 groups. (d, e) TUNEL assay indicated that positive nuclei were increased markedly in the OA synovial tissue compared with the sham-treated group. Positive cells were reduced in the Ac-YVAD-cmk and MCC950 groups. Data in this figure are presented as mean ± SD and analyzed by parametric test. Statistical significance is determined with one-way ANOVA followed by Bonferroni's post hoc test. ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001.

Article Snippet: The NLRP3 inhibitor MCC950 (MedChemExpress, Monmouth, NJ, USA) was diluted to 2 mg/ml with sterile saline and injected 10 mg/kg intraperitoneally.

Techniques: Western Blot, TUNEL Assay

Journal: Mediators of Inflammation

Article Title: Targeting NLRP3 Inflammasome Alleviates Synovitis by Reducing Pyroptosis in Rats with Experimental Temporomandibular Joint Osteoarthritis

doi: 10.1155/2022/2581151

Figure Lengend Snippet: LPS+ATP-treated macrophages. (a) Altered morphology of THP-1 cells under PMA stimulation. Scale bar = 50 μ m. (b, c) Western blotting results of NLRP3, ASC, cleaved caspase-1, IL-1 β , and GSDMD changes on protein levels in LPS-treated macrophages. (d) The results of qRT-PCR showed changes on mRNA levels among the control, LPS+ATP, LPS+ATP+MCC950, and LPS+ATP+AYC groups. Data are presented as mean ± SD and analyzed with one-way ANOVA followed by Bonferroni's post hoc test. ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001.

Article Snippet: The NLRP3 inhibitor MCC950 (MedChemExpress, Monmouth, NJ, USA) was diluted to 2 mg/ml with sterile saline and injected 10 mg/kg intraperitoneally.

Techniques: Western Blot, Quantitative RT-PCR, Control

Journal: Mediators of Inflammation

Article Title: Targeting NLRP3 Inflammasome Alleviates Synovitis by Reducing Pyroptosis in Rats with Experimental Temporomandibular Joint Osteoarthritis

doi: 10.1155/2022/2581151

Figure Lengend Snippet: LPS+ATP stimulated NLRP3 inflammasome- and pyroptosis-related molecules in the cultured FLS cells alone and cocultured system. (a, b) When FLS cells were cultured alone, lysate was collected for western blot after being treated with LPS+ATP. (c) Total RNAs of FLS cells were extracted for detecting mRNAs of NLRP3, ASC, caspase-1, GSDMD, and IL-1 β . Statistical analysis is run by independent samples t -test. (d, e) Secretion of IL-1 β and IL-18 in the supernate increased when FLS cell was cocultured with active THP-1 cell. (f–h) Western blotting and qRT-PCR results of cell lysate indicated the growing expression of NLRP3, ASC, caspase-1, GSDMD, and IL-1 β on protein and mRNA levels. Both the upregulation trend of the above tests after LPS+ATP intervention could be inhibited by MCC950 and AYC in the coculture system. Data are presented as mean ± SD; one-way ANOVA followed by Bonferroni's post hoc test was adopted for analysis. ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001.

Article Snippet: The NLRP3 inhibitor MCC950 (MedChemExpress, Monmouth, NJ, USA) was diluted to 2 mg/ml with sterile saline and injected 10 mg/kg intraperitoneally.

Techniques: Cell Culture, Western Blot, Quantitative RT-PCR, Expressing

Journal: Mediators of Inflammation

Article Title: Targeting NLRP3 Inflammasome Alleviates Synovitis by Reducing Pyroptosis in Rats with Experimental Temporomandibular Joint Osteoarthritis

doi: 10.1155/2022/2581151

Figure Lengend Snippet: Detection of pyroptosis in fibroblast-like synovial cells of coculture system. Cocultured FLS cells were challenged with LPS for 12 h and ATP for 2 h. Cell exposed to saline was as the control group. The MCC950 group and Ac-YVAD-cmk group were simultaneously added inhibitors to FLS cells. (a) PI- and Hoechst 33342-stained FLS cells in the control and LPS+ATP groups. (b) Quantification of PI-positive cells. Independent samples t -test. (c) The release of LDH was measured among the control, LPS+ATP, LPS+ATP+MCC950, and LPS+ATP+AYC groups. (d, e) Cells double stained by PI and kFluor488 for flow cytometry. One-way ANOVA followed by Bonferroni's post hoc test was used. ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001. (f) Representative transmission electron micrographs of FLS cells. Compared with control group, FLS cells exhibited pyroptotic features: extensive membrane bubbles and pores (red arrow); chromatin margination and condensation (yellow arrow); and swollen mitochondria with collapsed cristae (blue arrow).

Article Snippet: The NLRP3 inhibitor MCC950 (MedChemExpress, Monmouth, NJ, USA) was diluted to 2 mg/ml with sterile saline and injected 10 mg/kg intraperitoneally.

Techniques: Saline, Control, Staining, Flow Cytometry, Transmission Assay, Membrane

Journal: The EMBO Journal

Article Title: Poxvirus dsDNA genomes differentially activate AIM2 or NLRP3 inflammasomes in human primary cells

doi: 10.1038/s44318-025-00690-z

Figure Lengend Snippet: ( A – C ) Human monocyte-derived macrophages (hMDMs) differentiated in GM-CSF, ( D – F ) normal human epidermal keratinocytes (NHEK), ( G – I ) CD14 + monocytes, and ( J ) PMA-differentiated THP-1 macrophages were left untreated or pretreated with IFN-γ overnight. Cells were infected with indicated recombinant VACV strains at an MOI of 5 ( A – F , J ) or MOI 10 ( G – I ), in the presence ( A , C , D , F , G , I , J ) or absence ( B , E , H ) of VX, and where indicated, in the presence of 2.5 µM CRID3. Cells were harvested and fixed 6 h post infection. Infection (EGFP + cells) and C1C-EGFP speck assembly in infected or mock-treated cells was quantified by flow cytometry ( A , D , G , J ); infection data (EGFP + cells) corresponding to the experiment shown in panel ( J ) is displayed in Fig. . Membrane permeabilization was quantified by the uptake of DNA dye DRAQ7 over 6 h in an Incucyte live-cell imaging system ( B , E , H ). Scale bar = 100 μm. Samples for confocal microscopy were fixed and stained with 5 μg/mL wheat germ agglutinin (WGA)-Alexa Fluor (AF) 647 and 4 μM Hoechst 33342 ( C , F , I ). Scale bar = 5 μm. ( K ) PMA-differentiated WT or indicated knockout THP-1 cells were left untreated or pretreated with IFN-γ overnight and infected with the indicated reporter VACV strains at an MOI of 5 in the presence of VX. C1C-EGFP speck assembly was quantified as before. The corresponding infection data (EGFP + cells) is shown in Fig. . ( L , M ) NHEK ( L ) or PMA-differentiated THP-1 cells ( M ) constitutively expressing C1C-EGFP were left untreated or pretreated with IFN-γ overnight and infected with VACV WT or monkeypox virus (MPXV) at an MOI of 5, and where indicated, in the presence of CRID3. Cells were harvested and fixed 6 h post infection. VACV- or MPXV-infected cells were stained with rabbit polyclonal anti-H5 serum and AF647-labeled goat anti-rabbit IgG antibodies, and measured by flow cytometry. Corresponding infection data (αH5-AF647 + cells) is shown in Fig . C1C-EGFP speck assembly was analyzed in infected or mock-treated cells. Data represent average values (with individual data points) from N = 3 independent donors (primary cells) ± SEM, or from N = 3 independent experiments ± SEM (THP-1 cells). .

Article Snippet: CRID3 (MCC950) , Tocris , 5479.

Techniques: Derivative Assay, Infection, Recombinant, Flow Cytometry, Membrane, Live Cell Imaging, Confocal Microscopy, Staining, Knock-Out, Expressing, Virus, Labeling

Journal: The EMBO Journal

Article Title: Poxvirus dsDNA genomes differentially activate AIM2 or NLRP3 inflammasomes in human primary cells

doi: 10.1038/s44318-025-00690-z

Figure Lengend Snippet: ( A – I ) Human monocyte-derived macrophages (hMDMs) differentiated in GM-CSF ( A , D , E ), normal human epidermal keratinocytes (NHEK) ( B , F , G ), or human primary CD14 + monocytes ( C , H , I ), were left untreated or pretreated with IFN-γ overnight. Cells were infected with VACV C1C-EGFP at an MOI of 5 ( A , B ) or MOI 10 ( C ), in the absence of VX, and where indicated in the presence of 2.5 µM CRID3. Membrane permeabilization was quantified by the uptake of DNA dye DRAQ7 over 6 h in an Incucyte Live-Cell Imaging system ( A – C ). DRAQ7 uptake normalized to cell confluency are shown. IL-1β in the supernatant was measured by homogeneous time resolved fluorescence (HTRF) ( D , F , H ). Cell death was measured by LDH release and normalized to cells lysed in 1% Triton X-100 ( E , G , I ). ( J , K ) PMA-differentiated WT ( J ) or indicated knockout THP-1 cells ( K ) were left untreated or pretreated with IFN-γ overnight and infected with indicated VACV strains in the presence of VX, and where indicated, CRID3. Infection levels are displayed here; specking data from the same experiments are shown in Fig. . ( L , M ) NHEK ( L ) and PMA-differentiated THP-1 cells ( M ) constitutively expressing C1C-EGFP were left untreated or pretreated with IFN-γ overnight and infected with VACV or monkeypox virus (MPXV) at an MOI of 5 in the presence of VX, and where indicated, CRID3. Cells were fixed 6 h post infection and stained with rabbit polyclonal anti-H5 serum and AF647-labeled goat anti-rabbit IgG antibodies. Infected cells (EGFP + ) were analyzed in infected or mock-treated cells by flow cytometry as described before. Infection levels are displayed here; specking data from the same experiments are displayed in Fig. . Data represent average values (with individual data points) from N = 3 independent donors (primary cells) ± SEM, or from N = 3 independent experiments ± SEM (THP-1 cells).

Article Snippet: CRID3 (MCC950) , Tocris , 5479.

Techniques: Derivative Assay, Infection, Membrane, Live Cell Imaging, Fluorescence, Knock-Out, Expressing, Virus, Staining, Labeling, Flow Cytometry

Journal: The EMBO Journal

Article Title: Poxvirus dsDNA genomes differentially activate AIM2 or NLRP3 inflammasomes in human primary cells

doi: 10.1038/s44318-025-00690-z

Figure Lengend Snippet: ( A ) Genome structure of recombinant VACV strains expressing C1C-EGFP from the J2R early promoter (pE) and bivalent nanobodies from a synthetic early/late promoter (pE/L). Transgenes were inserted into the VACV TK locus by homologous recombination. ( B ) Scheme of AIM2 inflammasome components indicating the VACV-encoded nanobodies used to perturb inflammasome activation. ( C – E ) Human MDMs differentiated with GM-CSF ( C ), NHEKs ( D ), and CD14 + monocytes ( E ) were left untreated or treated with IFN-γ overnight. Cells were infected with VACV C1C-EGFP expressing the indicated nanobodies at an MOI of 5 ( C , D ) or 10 ( E ), in the presence of VX and, where indicated, CRID3. Six hours post infection, cells were harvested, fixed, and infection and C1C-EGFP speck assembly was analyzed by flow cytometry. Average values (with individual data points) from N = 3 independent donors ± SEM are displayed. .

Article Snippet: CRID3 (MCC950) , Tocris , 5479.

Techniques: Recombinant, Expressing, Homologous Recombination, Activation Assay, Infection, Flow Cytometry