m nlrp3 inhibitor mcc950  (MedChemExpress)

Journal: International Journal of Molecular Medicine

Article Title: Hypothermic machine perfusion protects DCD graft liver from ischemia-reperfusion injury by enhancing macrophage efferocytosis via KLF2-NLRP3 signaling

doi: 10.3892/ijmm.2026.5756

Figure Lengend Snippet: KLF2 inhibits the NLRP3-mediated pyroptosis pathway during CS/Rep in ECs. (A) Heatmap and (B) volcano plot of differentially expressed genes between control and ov-KLF2 HUVECs after CS/Rep treatment. mRNA levels of (C) KLF2 and (D) NLRP3 in ov-control and ov-KLF2 group of HUVECs following CS/Rep treatment were quantified using reverse transcription-quantitative PCR. (E) Co-IP analysis of the interaction between KLF2 and NLRP3. (F) Microstructure of HUVECS. (G) Representative Western blot images for KLF2, NLRP3, GSDMD, Caspase-1 and IL-18 in CS/Rep HUVECs. Quantitative analysis of KLF2 (H), NLRP3 (I), GSDMD (J), Caspase-1 (K) and IL-18 (L) protein expression based on western blot results from HUVECS of ov-control and ov-KLF2 groups. Quantitative analysis of KLF2 (M), NLRP3 (N), GSDMD (O), Caspase-1 (P) and IL-18 (Q) protein expression based on western blot results from HUVECS of sh-control and sh-KLF2 groups. (n=3). * P<0.05, ** P<0.01, *** P<0.001. KLF2, Kruppel-like Factor 2; CS/Rep, cold storage/reperfusion; HUVEC, human umbilical vein endothelial cells; ov, overexpression; IP, immunoprecipitation; GSDMD, gasdermin D; sh, short hairpin;; FC, fold-change.

Article Snippet: The incubation period between transfection and subsequent treatment was 72 h. To verify whether NLRP3 inhibition rescues the phenotypes resulting from KLF2 deficiency, sh-KLF2-transfected cells were treated (37°C) with 10 μ M NLRP3 inhibitor MCC950 (cat. no. HY12815, MedChemExpress) for 24 h and subjected to CS/Rep as aforementioned.

Techniques: Control, Reverse Transcription, Real-time Polymerase Chain Reaction, Co-Immunoprecipitation Assay, Western Blot, Expressing, Over Expression, Immunoprecipitation

Journal: The FASEB Journal

Article Title: Timing of Glucocorticoid Treatment Dictates Glucocorticoid Receptor Actions Modulating the NLRP3‐Inflammasome Activation in Macrophages

doi: 10.1096/fj.202504083R

Figure Lengend Snippet: GR actions over the repression of pro‐inflammatory genes requires a more accessible chromatin landscape in response to glucocorticoids in macrophages. (A) Volcano plot from RNA‐seq data in which each dot in red represents the differentially expressed genes (DEG) by Dex in LPS‐primed macrophages versus LPS‐treated with adjusted p < 0.05. The circle highlights a group of inflammasome‐associated genes. Schematic representation of the experimental setup for Dex treatment after LPS‐priming (B) and LPS plus Dex as a co‐treatment (co‐tx) (C). (D) Assessment of mRNA levels of Nlrp3, Nos2 , Acod1/Irg1, Il1b, Hif1a, P2ry2, Dusp1 , and Tsc22d3/Gilz genes in BMDM from Vehicle, Dex, LPS, Dex after LPS‐priming (after), and LPS plus Dex as a co‐treatment (co‐tx). The relative mRNA expression levels of the target genes were normalized to Ppib . (E) Immunoblot and densitometry analysis representing the levels of NLRP3, iNOS, ACOD1, and Pro‐IL1B in cell lysates from BMDMs, comparing the stimulation by 100 nM Dex at 6, 12, and 24 h in LPS‐primed macrophages (LPS → Dex) and as a cotreatment with LPS (LPS + Dex) as depicted in (B) and (C). (F) Meta‐profiles of GR signals over active TSSs and heatmaps depicting relative changes in Cut&Tag signal for GR enrichment in LPS, LPS plus Dex, and Dex after LPS treatments. Venn diagram illustrating the numbers of GR peaks in LPS plus Dex (LPS + Dex) and Dex after LPS (LPS → Dex) regimens. (G) Browser image of Cut&Tag signal for GR peaks over Per1 , Dusp1 , Ccl3 , Ccl4 , and Nlrp3 genes as induced and reduced by Dex treatment. (H) Frequency distribution graph for motifs enrichment analysis. The x ‐axis is the ratio of % GR peaks over % background regions, and the y ‐axis represents the −log10 of the p value. NHR: Nuclear hormone receptor. Data are mean ± SEM. Blots are representative of a minimum of 3 independent experiments. Statistical analysis was performed using one‐way ANOVA with Tukey's multiple‐comparison test (A) and (B). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: Lipopolysaccharide from E. coli 0111: B4 strain (LPS‐EB), NLRP3 inhibitor MCC950, Caspase‐1 inhibitor Ac‐Y‐VAD‐cmk, 4‐octyl‐itaconate (4‐OI), adenosine 5′‐triphosphate disodium salt (ATP), nigericin, and monosodium urate (MSU) crystals were purchased from InvivoGen (San Diego, CA, USA).

Techniques: RNA Sequencing, Expressing, Western Blot, Comparison

Journal: The FASEB Journal

Article Title: Timing of Glucocorticoid Treatment Dictates Glucocorticoid Receptor Actions Modulating the NLRP3‐Inflammasome Activation in Macrophages

doi: 10.1096/fj.202504083R

Figure Lengend Snippet: Glucocorticoids modulate inflammation and pyroptosis by regulating late NLRP3‐inflammasome activation through ACOD1 and iNOS. (A) Schematic representation of the experimental setup for conventional NLRP3‐inflammasome activation (upper), and late NLRP3‐inflammasome activation regulated by Dex and MCC950 (lower). (B) and (C) Quantification of IL‐1β concentration determined by ELISA in the supernatant of WT and GRKO BDMDs stimulated with LPS for 3 h (conventional) and 24 h (late), with Dex and vehicle added during the final 2 and 8 h of treatment, followed by ATP (5 mM) or Nigericin (10 μM) ( n = 3). (D) Immunoblot analysis depicting Dex effects on the protein levels of ACOD1, iNOS, NLRP3, pro‐IL‐1β, Caspase‐1 and GSDM‐D during late LPS‐induced NLRP3‐inflammasome activation and using ATP as signal 2 in WT and GRKO BMDMs lysates, detected with specific antibodies using western blotting. (E) Densitometry values of the immunoreactive bands quantified for protein content, normalized to β‐actin as depicted in (D) ( n = 4). (F) Nitrite levels in the supernatants of WT and GRKO BMDMs as depicted in (D) determined by colorimetric assay ( n = 4). (G) Assessment of lactate dehydrogenase (LDH) activity in the supernatants of WT and GRKO BMDMs as depicted in (D) ( n = 4). (H) Immunoblot analysis depicting the protein levels of NRF2 as depicted in (D) in presence of the proteasome inhibitor MG‐132 (2 μM) 6 h before harvesting cells ( n = 3). (I) Immunoblot analysis depicting the protein levels of HIF‐1α as depicted in (D) in presence of the prolyl‐hydroxylase inhibitor Roxadustat (RXD; 10 μM) 2 h before adding Dex ( n = 3). (J) Quantification of IL‐1β concentration determined by ELISA in the supernatant of WT BDMDs as depicted in (H) and (I) ( n = 3). (K) Assessment of lactate dehydrogenase (LDH) activity in supernatants of WT BMDMs subjected to the conditions described in (H) and (I) ( n = 3). Data represented as mean ± SEM. Blots shown are representative of a minimum of 3 independent experiments. Statistical analysis was performed using 2‐way ANOVA with Sidak's multiple‐comparison test (B–I); one‐way ANOVA with Tukey's multiple‐comparison test (J) and (K). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: Lipopolysaccharide from E. coli 0111: B4 strain (LPS‐EB), NLRP3 inhibitor MCC950, Caspase‐1 inhibitor Ac‐Y‐VAD‐cmk, 4‐octyl‐itaconate (4‐OI), adenosine 5′‐triphosphate disodium salt (ATP), nigericin, and monosodium urate (MSU) crystals were purchased from InvivoGen (San Diego, CA, USA).

Techniques: Activation Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Colorimetric Assay, Activity Assay, Comparison

Journal: The FASEB Journal

Article Title: Timing of Glucocorticoid Treatment Dictates Glucocorticoid Receptor Actions Modulating the NLRP3‐Inflammasome Activation in Macrophages

doi: 10.1096/fj.202504083R

Figure Lengend Snippet: Clinically relevant course of glucocorticoid plays a role in suppressing sustained NLRP3‐Inflammasome activation. (A) Assessment of mRNA levels of Nos2 , Acod1/Irg1, Il1b, Nlrp3, Hif1a, Nrf2, P2ry2 , Drp1, Mfn2 , and Dusp1 genes during late LPS‐induced NLRP3‐inflammasome activation and using ATP as signal 2 in WT BDMDs using Dex after LPS‐priming (blue) and LPS plus Dex as a co‐treatment (red). (B) Immunoblot analysis depicting Dex effects when is added after LPS priming (LPS → Dex) or as a co‐treatment (LPS + Dex) on the protein levels of ACOD1, iNOS, NLRP3, DRP‐1, MFN2, NT‐GSDMD, pro‐IL‐1β and DUSP1/MKP1 during the late LPS‐induced NLRP3‐inflammasome activation post ATP. (C) Quantification of IL‐1β determined by ELISA in the supernatant of BMDMs as depicted in (B). (D) Assessment of lactate dehydrogenase (LDH) activity in the supernatants of BMDMs as depicted in (B) ( n = 3). (E) Nitrite levels determined by colorimetric assay in the supernatants of BMDMs as depicted in (B) ( n = 4). Data represented as mean ± SEM. Blots shown are representative of a minimum of 3 independent experiments. Statistical analysis was performed using one‐way ANOVA with Tukey's multiple‐comparison test for (A) and (C–E) and 2‐way ANOVA with Sidak's multiple‐comparison test (B). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: Lipopolysaccharide from E. coli 0111: B4 strain (LPS‐EB), NLRP3 inhibitor MCC950, Caspase‐1 inhibitor Ac‐Y‐VAD‐cmk, 4‐octyl‐itaconate (4‐OI), adenosine 5′‐triphosphate disodium salt (ATP), nigericin, and monosodium urate (MSU) crystals were purchased from InvivoGen (San Diego, CA, USA).

Techniques: Activation Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Activity Assay, Colorimetric Assay, Comparison

Journal: The FASEB Journal

Article Title: Timing of Glucocorticoid Treatment Dictates Glucocorticoid Receptor Actions Modulating the NLRP3‐Inflammasome Activation in Macrophages

doi: 10.1096/fj.202504083R

Figure Lengend Snippet: The loss of Glucocorticoid Receptor (GR) in murine myeloid cells and the block of GR signaling in human monocytes‐derived macrophages exacerbates the NLRP3 inflammasome activation. (A) Schematic representation of the experimental setup for in vivo peritonitis model using MSU crystal. Peritoneal lavage fluid was collected 6 h post‐ intraperitoneal injection of 30 mg/kg MSU crystals in WT and myeloid‐GRKO mice. Peritoneal lavage involved the injection of 2.5 mL PBS and subsequent retrieval from the peritoneal cavity for supernatant harvesting and immune cells isolation. (B) Evaluation of IL‐1β concentration in peritoneal lavage. (C) Determination of absolute numbers of infiltrating neutrophils in peritoneal lavage, as was described in (A), calculated based on cell count per mL and the total recovered volume. (D) Relative percentage of small peritoneal macrophages (SPM) and (E) large peritoneal macrophages (LPM) expressing differentially the markers F4/80 and MHC‐II. These cell populations were gated from CD11b + /CD115 + cells. Data represented as mean ± SEM ( n = 9, from B to E, where each dot representing one mouse, encompassing both female and male). (F) Schematic representation of the experimental setup for late NLRP3‐inflammasome activation in human monocytes‐derived macrophages (MDMs) modulated by Dex in the presence of the GR antagonist RU‐486. (G) Quantification of NLRP3 normalized to β‐actin in lysates from human MDMs stimulated with LPS for 24 h, with the addition of the antagonist RU‐486 and Dex during the final 8 h, followed by ATP (5 mM). (H) Quantification of ACOD1 protein as depicted in (G). (I) Quantification of pro‐IL‐1β protein as depicted in (G). (J) Assessment of IL‐1β secretion in human MDMs stimulated as described in (G), determined by ELISA ( N = 6). Data representative of 4–6 healthy donors. Statistical analysis was performed using two‐tailed unpaired t ‐tests (B–E) and one‐way ANOVA with Tukey's multiple‐comparison test (G–J) * p < 0.05; ** p < 0.01.

Article Snippet: Lipopolysaccharide from E. coli 0111: B4 strain (LPS‐EB), NLRP3 inhibitor MCC950, Caspase‐1 inhibitor Ac‐Y‐VAD‐cmk, 4‐octyl‐itaconate (4‐OI), adenosine 5′‐triphosphate disodium salt (ATP), nigericin, and monosodium urate (MSU) crystals were purchased from InvivoGen (San Diego, CA, USA).

Techniques: Blocking Assay, Derivative Assay, Activation Assay, In Vivo, Injection, Isolation, Concentration Assay, Cell Characterization, Expressing, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Comparison

Journal: Journal of Neuroinflammation

Article Title: PKM2 preconditioning protects endothelial cells from pyroptosis and BBB disruption via NRF2/TRX/TXNIP signaling in neonatal hypoxic-ischemic brain injury

doi: 10.1186/s12974-026-03694-6

Figure Lengend Snippet: Pyroptosis disrupts BBB integrity by upregulating the expression of MMP2 and MMP9. Rats were intraperitoneally pretreated with the NLRP3 activator nigericin (4 mg/kg/day) 2 h before HI injury. A , B Protein levels ( A ) and quantitative analysis ( B ) of the junction proteins ZO-1, P120, β-catenin, and occludin in rats ( n = 5). C , D Images ( C ) and quantitative analysis ( D ) of ZO-1 using double staining for ZO-1 (green) and CD31 (red) in rat brains ( n = 5; scale bar = 50 μm). E , F Images of Evans blue-stained brains ( F ) and quantification ( E ) of Evans blue content in each group of rats ( n = 5). G FITC-dextran extravasation in rats ( n = 5). (H) Protein levels and quantification of MMP9 and MMP2 in rats ( n = 5)

Article Snippet: Either the NLRP3 activator nigericin (MedChemExpress, Catalog #HY-127019, 4 mg/kg/day) or the caspase-1 inhibitor Ac-YVAD (Sigma–Aldrich, Catalog #SML0429, 1 mg/kg/day) was injected intraperitoneally 2 h before HI injury.

Techniques: Expressing, Double Staining, Staining

Journal: Journal of Neuroinflammation

Article Title: PKM2 preconditioning protects endothelial cells from pyroptosis and BBB disruption via NRF2/TRX/TXNIP signaling in neonatal hypoxic-ischemic brain injury

doi: 10.1186/s12974-026-03694-6

Figure Lengend Snippet: PKM2 inhibition upregulates pyroptosis in HI-treated endothelial cells in vivo. Rats were intraperitoneally pretreated with the PKM2 inhibitor Compound 3k (5 mg/kg/day) alone or together with the NLRP3 inhibitor MCC950 (10 mg/kg/day) 2 h before HI injury. A , B Representative western blot bands ( A ) and quantitative analysis ( B ) of pyroptosis-related proteins in rats ( n = 5). C , D Graphs of immunofluorescence staining ( C ) and quantitative analysis ( D ) of caspase-1 (green) colocalization on endothelial cells (CD31, red) in rats ( n = 5; scale bar = 50 μm). E LDH release was assessed in rats after HI injury ( n = 5)

Article Snippet: Either the NLRP3 activator nigericin (MedChemExpress, Catalog #HY-127019, 4 mg/kg/day) or the caspase-1 inhibitor Ac-YVAD (Sigma–Aldrich, Catalog #SML0429, 1 mg/kg/day) was injected intraperitoneally 2 h before HI injury.

Techniques: Inhibition, In Vivo, Western Blot, Immunofluorescence, Staining

Journal: Journal of Neuroinflammation

Article Title: PKM2 preconditioning protects endothelial cells from pyroptosis and BBB disruption via NRF2/TRX/TXNIP signaling in neonatal hypoxic-ischemic brain injury

doi: 10.1186/s12974-026-03694-6

Figure Lengend Snippet: PKM2 overexpression promotes NRF2 expression to inhibit TXNIP and pyroptosis in hCMECs. HCMECs were pretreated with the NRF2 inhibitor ML385 (20 µM) for 2 h alone or together with the recombinant plasmid Flag-PKM2 for 6 h, followed by OGD for an additional 18 h. A , E , G Protein levels ( A ) and quantification ( E , G ) of NRF2 in the cytoplasm and TXNIP, HO-1, TRX, and NLRP3 in hCMECs ( n = 5). B , C Western blot ( B ) and quantification ( C ) for nuclear NRF2 in hCMECs ( n = 5). D Immunoprecipitation with anti-NLRP3 or anti-TXNIP from control and OGD-treated hCMECs. F , I Immunofluorescence images ( F ) and quantification ( I ) of NRF2 expression 18 h after OGD ( n = 5; scale bar = 50 μm). H , J Immunofluorescence images ( H ) and quantification ( J ) of HO-1 expression 18 h after OGD ( n = 5; scale bar = 50 μm)

Article Snippet: Either the NLRP3 activator nigericin (MedChemExpress, Catalog #HY-127019, 4 mg/kg/day) or the caspase-1 inhibitor Ac-YVAD (Sigma–Aldrich, Catalog #SML0429, 1 mg/kg/day) was injected intraperitoneally 2 h before HI injury.

Techniques: Over Expression, Expressing, Recombinant, Plasmid Preparation, Western Blot, Immunoprecipitation, Control, Immunofluorescence

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Sepsis-induced acute lung injury: AUF1 regulates pyroptosis via ETS2/ZDHHC21-mediated STING palmitoylation

doi: 10.1007/s00018-025-06038-4

Figure Lengend Snippet: Assessment of survival, lung pathology, STING palmitoylation, and inflammatory markers in sepsis-induced acute lung injury. The cecal ligation and puncture (CLP) model was utilized to create sepsis-related acute lung injury (ALI). Samples were harvested for analysis 72 h after CLP. The NLRP3/AIM2-IN-3 group received an intravenous injection through the tail vein immediately after CLP induction at a dose of 10 mg/kg. ( A ) Survival rates were compared using Kaplan-Meier survival curves among Sham-operated mice, CLP-induced sepsis mice, and CLP-induced sepsis mice treated with the NLRP3/AIM2 inhibitor NLRP3/AIM2-IN-3 (CLP + NLRP3/AIM2-IN-3). ( B ) Lung tissues from Sham, CLP, and CLP + NLRP3/AIM2-IN-3 groups were subjected to hematoxylin and eosin (HE) staining for assessment of pathological changes (scale bar = 100 μm). ( C ) Lung injury was quantitatively assessed based on histological criteria, providing injury scores for Sham, CLP, and CLP + NLRP3/AIM2-IN-3 groups. ( D ) STING palmitoylation levels in lung tissues were measured using the Acyl-resin-assisted capture (Acyl-RAC) assay in Sham, CLP, and CLP + NLRP3/AIM2-IN-3 groups. ( E ) Lung tissue sections underwent immunohistochemistry (IHC) staining to assess the expression of ZDHHC21, cleaved Caspase-1 (c-Caspase-1), and the cleaved form of Gasdermin D (GSDMD-N) in Sham, CLP, and CLP + NLRP3/AIM2-IN-3 groups (scale bar = 100 μm). ( F ) Protein levels of ZDHHC21,TBK1, p-TBK1, IRF3, p-IRF3, c-Caspase-1, and GSDMD-N were quantified in lung tissues from Sham, CLP, and CLP + NLRP3/AIM2-IN-3 groups using Western blotting. ( G ) Levels of the inflammatory cytokines IL-18 and IL-1β in lung tissues were measured by enzyme-linked immunosorbent assay (ELISA) in Sham, CLP, and CLP + NLRP3/AIM2-IN-3 groups. Data are presented as mean ± SD. Statistical significance was determined using one-way ANOVA followed by Tukey’s post hoc test. n = 8 mice per group. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: CLP + NLRP3/AIM2-IN-3 Group: CLP-induced sepsis mice received intraperitoneal injections of the NLRP3/AIM2 inhibitor NLRP3/AIM2-IN-3 (Cat: HY-144226, MedChemExpress, NJ, USA) at a dose of 10 mg/kg immediately after surgery.

Techniques: Ligation, Injection, Staining, Immunohistochemistry, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Sepsis-induced acute lung injury: AUF1 regulates pyroptosis via ETS2/ZDHHC21-mediated STING palmitoylation

doi: 10.1007/s00018-025-06038-4

Figure Lengend Snippet: Evaluation of cell viability, pyroptosis, STING palmitoylation, and inflammatory markers in an in vitro sepsis-induced acute lung injury model. MLE-12 cells were stimulated with LPS (1 µg/mL) to induce a sepsis-induced ALI cell model, with the control group receiving an equivalent volume of PBS. All assays were performed 24 h after LPS stimulation. The NLRP3/AIM2-IN-3 group was treated with 10 µM of NLRP3/AIM2-IN-3 1 h prior to LPS stimulation. ( A ) The CCK-8 assay was conducted to assess the cell viability in control, LPS, and LPS + NLRP3/AIM2-IN-3 treated MLE-12 cells. ( B ) Proportions of PI-positive and Caspase-1-positive cells were analyzed by flow cytometry in control, LPS, and LPS + NLRP3/AIM2-IN-3 treated MLE-12 cells. ( C ) STING palmitoylation levels were assessed using the Acyl-resin-assisted capture (Acyl-RAC) assay in control, LPS, and LPS + NLRP3/AIM2-IN-3 treated MLE-12 cells. ( D ) ZDHHC21, TBK1, p-TBK1, IRF3, p-IRF3, cleaved Caspase-1 (c-Caspase-1), and GSDMD-N protein expression were determined by Western blot analysis in control, LPS, and LPS + NLRP3/AIM2-IN-3 treated MLE-12 cells. ( E ) IL-1β and IL-18 concentrations in the cell culture supernatant were measured by ELISA in control, LPS, and LPS + NLRP3/AIM2-IN-3 treated MLE-12 cells. Data are presented as mean ± SD. Statistical significance was assessed using one-way ANOVA followed by Tukey’s post hoc test. n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: CLP + NLRP3/AIM2-IN-3 Group: CLP-induced sepsis mice received intraperitoneal injections of the NLRP3/AIM2 inhibitor NLRP3/AIM2-IN-3 (Cat: HY-144226, MedChemExpress, NJ, USA) at a dose of 10 mg/kg immediately after surgery.

Techniques: In Vitro, Control, CCK-8 Assay, Flow Cytometry, Expressing, Western Blot, Cell Culture, Enzyme-linked Immunosorbent Assay