Journal: Redox Biology

Article Title: Neutrophil extracellular traps exacerbate inflammatory arthritis by inhibiting γδ Treg cell differentiation via the AIM2 inflammasome

doi: 10.1016/j.redox.2025.103881

Figure Lengend Snippet: NET-associated DNA activated the AIM2 inflammasome to inhibit γδ Treg cell differentiation A. Principal component analysis of RNA-seq data from the NC and NET groups (n = 3). B. Volcano plots of the RNA-seq data from both the NC and NET groups (n = 3). C. Cluster heatmap of differentially expressed genes identified via RNA-seq in the NC and NET groups (n = 3). D. KEGG analysis of RNA-seq data from the NC and NET groups (n = 3). E. Relative protein levels of AIM2, FL-GSDMD, NT-GSDMD, Caspase-1 and cleaved Caspase-1 in γδ T cells stimulated with or without NETs, NETs treated with DNAse or NET-associated DNA (n = 6). F. Efficiency of AIM2 knockdown in γδ T cells via lentivirus transfection (n = 5). G. γδ T cells were transfected with an AIM2-knockdown lentivirus or control vector; subsequently, the cells were cultured in the presence or absence of NETs. The proportion of γδ Treg cells among the total γδ T cells was analyzed by flow cytometry (n = 5). H. γδ T cells isolated from wild-type (WT) or AIM2-deficient (AIM2-/-) mice were polarized under γδ Treg conditions with or without NETs stimulation. The proportion of γδ Treg cells was analyzed by flow cytometry (n = 6). The data are presented as the means ± SEMs. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ns not significant, p > 0.05.

Article Snippet: For the indicated experiments, NETs, NET-associated DNA, a CIE inhibitor (Selleck, #S9670), an AIM2 inhibitor (MCE, #HY-144226), an AIM2 activator (Poly(dA:dT); MCE, #HY-138646), N-Acetyl- l -cysteine (1 mM, MCE, #HY-B0215), Tempo (10 nM, MCE, #HY-W001187) and Disulfiram (MCE, # HY-B0240) were added.

Techniques: Cell Differentiation, RNA Sequencing, Knockdown, Transfection, Control, Plasmid Preparation, Cell Culture, Flow Cytometry, Isolation

Journal: Redox Biology

Article Title: Neutrophil extracellular traps exacerbate inflammatory arthritis by inhibiting γδ Treg cell differentiation via the AIM2 inflammasome

doi: 10.1016/j.redox.2025.103881

Figure Lengend Snippet: AIM2 inflammasome activation induced ROS accumulation A-C. γδ T cells were transfected with an AIM2-knockdown lentivirus or control vector; subsequently, the cells were cultured in the presence or absence of NETs. A. γδ T cells were stained with CellROX (red) and live cell nuclear stain Hoechst33342. Mean fluorescence intensity (MFI) measured using ImageJ software. B. As in (A) but with the mitochondrial-targeted superoxide dye MitoSOX. Scale bar = 20 μm. C. Total cellular ROS levels and mtROS levels of γδ T cells and quantitative analysis of the MFI of ROS and mtROS (n = 6). D-F. γδ T cells were treated with N-acetylcysteine (NAC) or Mito-TEMPO (mtT); subsequently, the cells were cultured in the presence or absence of NETs. D. Total cellular ROS levels of γδ T cells and quantitative analysis of the MFI of ROS (n = 6). E. mtROS levels of γδ T cells and quantitative analysis of the MFI mtROS (n = 6). F. The proportion of γδ Treg cells among the total γδ T cells was analyzed by flow cytometry (n = 6). The data are presented as the means ± SEMs. ∗∗∗p < 0.001; ns not significant p > 0.05.

Article Snippet: For the indicated experiments, NETs, NET-associated DNA, a CIE inhibitor (Selleck, #S9670), an AIM2 inhibitor (MCE, #HY-144226), an AIM2 activator (Poly(dA:dT); MCE, #HY-138646), N-Acetyl- l -cysteine (1 mM, MCE, #HY-B0215), Tempo (10 nM, MCE, #HY-W001187) and Disulfiram (MCE, # HY-B0240) were added.

Techniques: Activation Assay, Transfection, Knockdown, Control, Plasmid Preparation, Cell Culture, Staining, Fluorescence, Software, Flow Cytometry

Journal: Redox Biology

Article Title: Neutrophil extracellular traps exacerbate inflammatory arthritis by inhibiting γδ Treg cell differentiation via the AIM2 inflammasome

doi: 10.1016/j.redox.2025.103881

Figure Lengend Snippet: NET-mediated AIM2 inflammasome induced mitochondrial dysfunction γδ T cells were transfected with an AIM2-knockdown lentivirus or control vector; subsequently, the cells were cultured in the presence or absence of NETs. A. Mitochondria (MitoTracker-red; red) in γδ T cells. Nuclei (DAPI; blue). Scale bar = 5 μm. B. Relative protein levels of p-Drp1 (n = 6). C. Mitochondrial membrane potential was determined by JC-1 staining. Hoechst33342 was used for nuclear staining. D. The ratio of aggregates/monomers (n = 6). E. Mitochondrial membrane potential measured by flow cytometry of JC-1. F. The ratio of high mitochondrial potential (n = 6). G. Mitochondrial membrane potential measured by flow cytometry of TMRE. Quantitation is on the right (n = 6). H. Curve and quantitative analysis of the OCRs for γδ T cells (n = 5). The data are presented as the means ± SEMs. ∗p < 0.05 ; ∗∗∗p < 0.001 ; ns not significant p > 0.05.

Article Snippet: For the indicated experiments, NETs, NET-associated DNA, a CIE inhibitor (Selleck, #S9670), an AIM2 inhibitor (MCE, #HY-144226), an AIM2 activator (Poly(dA:dT); MCE, #HY-138646), N-Acetyl- l -cysteine (1 mM, MCE, #HY-B0215), Tempo (10 nM, MCE, #HY-W001187) and Disulfiram (MCE, # HY-B0240) were added.

Techniques: Transfection, Knockdown, Control, Plasmid Preparation, Cell Culture, Membrane, Staining, Flow Cytometry, Quantitation Assay

Journal: Redox Biology

Article Title: Neutrophil extracellular traps exacerbate inflammatory arthritis by inhibiting γδ Treg cell differentiation via the AIM2 inflammasome

doi: 10.1016/j.redox.2025.103881

Figure Lengend Snippet: AIM2 inflammasome effector GSDMD induced mitochondrial dysfunction and ROS accumulation A-B. γδ T cells were transfected with an AIM2-knockdown lentivirus or control vector; subsequently, the cells were cultured in the presence or absence of NETs. A. GSDMD association (anti-GSDMD) with the mitochondrial network measured by flow cytometry of isolated mitochondria (n = 6). B. N-GSDMD mitochondrial association in γδ T cells via biochemical fractionation and immunoblot, with VDAC1 to control for mitochondrial and with GAPDH to control for cytoplasm (n = 6). C–F. γδ T cells were treated with Disulfiram (DSF), and the cells were cultured in the presence or absence of NETs. C. Mitochondrial membrane potential measured by flow cytometry of TMRE (n = 6). D. Total cellular ROS levels and mtROS levels of γδ T cells and quantitative analysis of the MFI of ROS and mtROS (n = 6). E. Curve and quantitative analysis of the OCRs for γδ T cells (n = 5). F. The proportion of γδ Treg cells among the total γδ T cells was analyzed by flow cytometry (n = 6). The data are presented as the means ± SEMs. ∗p < 0.05 ; ∗∗∗p < 0.001 ; ns not significant p > 0.05.

Article Snippet: For the indicated experiments, NETs, NET-associated DNA, a CIE inhibitor (Selleck, #S9670), an AIM2 inhibitor (MCE, #HY-144226), an AIM2 activator (Poly(dA:dT); MCE, #HY-138646), N-Acetyl- l -cysteine (1 mM, MCE, #HY-B0215), Tempo (10 nM, MCE, #HY-W001187) and Disulfiram (MCE, # HY-B0240) were added.

Techniques: Transfection, Knockdown, Control, Plasmid Preparation, Cell Culture, Flow Cytometry, Isolation, Fractionation, Western Blot, Membrane

Journal: Redox Biology

Article Title: Inhibition of NLRP3-mediated crosstalk between hepatocytes and liver macrophages by geniposidic acid alleviates cholestatic liver inflammatory injury

doi: 10.1016/j.redox.2022.102404

Figure Lengend Snippet: GPA directly binds to NLRP3 and represses its expression. (A) Dual-luciferase reporter assay results. RAW264.7 cells were transiently co‐transfected with NLRP3-luc plasmids for 12 h and treated with DMSO (vehicle control), GPA-H (100 μM), GPA-M (50 μM), GPA-L (25 μM), or MCC950 (10 μM) for 24 h. Relative luciferase activity was calculated by ration of firefly luciferase/renilla luciferase activity (n = 5). * P < 0.05, ** P < 0.01, compared to the DMSO control transfected with empty vector. Data are expressed as the mean ± SEM of 5 independent experiments ( n = 5). (B) mRNA levels of NLRP3, NFκB1, and IL-1β in THP-1 monocyte-derived macrophages and BMDM as analyzed by real-time PCR after 24 h treatment with different concentrations of GPA (25–100 μM). Results are expression as fold changes compared to the DMSO group. Data are expressed as the mean ± SEM of 5 independent experiments ( n = 5). * P < 0.05, ** P < 0.01 versus DMSO control group. (C) Protein levels of NLRP3, ASC, CASP-1, and IL-1β in THP-1 monocyte-derived macrophages were determined by Western blot analysis after 24 h treatment with different concentrations of GPA. β-ACTIN was used as internal reference protein. Data are expressed as the mean ± SEM of 3 independent experiments( n = 3). (D) Cell lysates of LPS-primed THP-1 monocytes were incubated with GPA (100 μM) or Bio-GPA (100 μM) for 4 h and subjected to pull-down assays with streptavidin beads. Total (input), bound (pull-down), and remaining proteins (after pull-down) were immunoblotted as indicated. β-ACTIN was used as internal reference protein.Data are expressed as the mean ± SEM of 3–4 independent experiments ( n = 3–4). (E) Competitive experiment. Cell lysates of LPS-primed THP-1 monocytes were incubated with Bio-GPA (100 μM) and different concentrations of free GPA (50 or 100 μM) and subjected to pull-down assays with streptavidin beads. β-ACTIN was used as internal reference protein. Data are expressed as the mean ± SEM of 3–4 independent experiments ( n = 3–4). (F) Human recombinant NLRP3 proteins were incubated with the indicated doses of Bio-GPA (50 or 100 μM) and subjected to pull-down assays with streptavidin beads. NLRP3 was used as internal reference protein. Data are expressed as the mean ± SEM of 3–4 independent experiments ( n = 3–4). (G) Human recombinant NLRP3 proteins were incubated with Bio-GPA (100 μM) or/and β-Mercaptoethanol (50 μM) and subjected to pull-down assays with streptavidin beads. NLRP3 was used as internal reference protein. Data are expressed as the mean ± SEM of 3–4 independent repeats ( n = 3–4).

Article Snippet: TaqMan probes, including those for human-specific NLRP3 (Hs00918082), NF κ B1 (Hs00765730), IL-1 β (Hs00174097), IL-6 (Hs00174131 ) , ICAM-1 (Hs00164932), COX-2 (Hs00153133), TNF- α (Hs00174128), and GAPDH (Hs02786624); and mouse-specific Nlrp3 (Mm00840904), Nf κ b1 (Mm00476361), Il-1 β (Mm00434228), Il-6 (Mm00446190 ) , Icam-1 (Mm00516023), Cox-2 (Mm00478374), Tnf- α (Mm00443258), Cyp7a1 (Mm00484150), Cyp8b1 (Mm00501637), Cyp27a1 (Mm00470430), Cyp2b10 (Mm01972453), Cyp3a11 (Mm00731567), Ugt1a1 (Mm02603337), Abcb11 (Mm00445168), Abcc2 (Mm00496899), Abcc3 (Mm00551550), Abcc4 (Mm01226381), Slc01b2 (Mm00451510), Slc10a1 (Mm00441421), and Gapdh (Mm99999915), were acquired from Invitrogen (Carlsbad, CA, USA).

Techniques: Expressing, Luciferase, Reporter Assay, Transfection, Control, Activity Assay, Plasmid Preparation, Derivative Assay, Real-time Polymerase Chain Reaction, Western Blot, Incubation, Recombinant

Journal: Redox Biology

Article Title: Inhibition of NLRP3-mediated crosstalk between hepatocytes and liver macrophages by geniposidic acid alleviates cholestatic liver inflammatory injury

doi: 10.1016/j.redox.2022.102404

Figure Lengend Snippet: GPA reduces LPS-induced NLRP3 expression via inhibiting NF-κB signaling. (A – C) THP-1 monocytes were pretreated with the indicated concentrations of GPA for 24 h and stimulated with LPS (1 μg/ml) for 24 h. The levels of mRNA (A) , protein (B) , and pro-inflammatory cytokines in supernatants (C) were measured by real-time PCR, Western blot, and ELISA, respectively. mRNA results are expressed as fold changes compared to the DMSO group.β-ACTIN was used as internal reference protein. Data are expressed as the mean ± SEM of 3–5 independent experiments ( n = 5, A and C; n = 3, B). # P < 0.05, ## P < 0.01 versus DMSO-treated control group, * P < 0.05, ** P < 0.01 versus LPS-treated group. (D – E) BMDM were pretreated with indicated concentrations of GPA for 24 h and stimulated with LPS (1 μg/ml) for 24 h. The levels of mRNA (D) and pro-inflammatory cytokines in supernatants (E) were determined by real-time PCR and ELISA, respectively. mRNA results are expressed as fold changes compared to the DMSO group. Data are expressed as the mean ± SEM of 5 independent experiments ( n = 5). # P < 0.05, ## P < 0.01 versus DMSO-treated control group, * P < 0.05, ** P < 0.01 versus LPS-treated group. (F and G) Nlrp3 −/− BMDM were pretreated with indicated concentrations of GPA for 24 h and stimulated with LPS (1 μg/ml) for 24 h. The levels of mRNA (F) and pro-inflammatory cytokines in supernatants (G) were determined by real-time PCR and ELISA, respectively. mRNA results are expressed as fold changes compared to the DMSO group. Data are expressed as the mean ± SEM of 5 independent experiments ( n = 5). # P < 0.05, ## P < 0.01 versus DMSO-treated control group, * P < 0.05, ** P < 0.01 versus LPS-treated group. (H) Dual-luciferase reporter assay results. RAW274.6 cells were transiently co‐transfected with NF-κB-luc plasmids for 12 h and treated with DMSO (vehicle control), GPA-H (100 μM), GPA-M (50 μM), GPA-L (25 μM), or MCC950 (10 μM) for 12 h, followed by 24 h induction with LPS (1 μg/ml). Data are expressed as the mean ± SEM of 5 independent experiments ( n = 5). (I and J) The protein expression levels of NF-κB (I), NLRP3 and ASC (J) in THP-1 monocytes were measured by immunofluorescence assay. The nuclei were stained with DAPI in blue, and targeted protein was stained in green or red (magnification: 630 × ). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: TaqMan probes, including those for human-specific NLRP3 (Hs00918082), NF κ B1 (Hs00765730), IL-1 β (Hs00174097), IL-6 (Hs00174131 ) , ICAM-1 (Hs00164932), COX-2 (Hs00153133), TNF- α (Hs00174128), and GAPDH (Hs02786624); and mouse-specific Nlrp3 (Mm00840904), Nf κ b1 (Mm00476361), Il-1 β (Mm00434228), Il-6 (Mm00446190 ) , Icam-1 (Mm00516023), Cox-2 (Mm00478374), Tnf- α (Mm00443258), Cyp7a1 (Mm00484150), Cyp8b1 (Mm00501637), Cyp27a1 (Mm00470430), Cyp2b10 (Mm01972453), Cyp3a11 (Mm00731567), Ugt1a1 (Mm02603337), Abcb11 (Mm00445168), Abcc2 (Mm00496899), Abcc3 (Mm00551550), Abcc4 (Mm01226381), Slc01b2 (Mm00451510), Slc10a1 (Mm00441421), and Gapdh (Mm99999915), were acquired from Invitrogen (Carlsbad, CA, USA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay, Control, Luciferase, Reporter Assay, Transfection, Immunofluorescence, Staining

Journal: Redox Biology

Article Title: Inhibition of NLRP3-mediated crosstalk between hepatocytes and liver macrophages by geniposidic acid alleviates cholestatic liver inflammatory injury

doi: 10.1016/j.redox.2022.102404

Figure Lengend Snippet: GPA suppresses NLRP3 inflammasome activation. (A – C) THP-1 monocytes were pretreated with indicated concentrations of GPA for 24 h, stimulated with LPS (1 μg/ml) for 24 h and ATP (2.5 mM) for 30 min. (A) The levels of pro-inflammatory cytokines in cell-culture supernatants, including IL-1β, IL-6, and TNF-α were quantified by ELISA. Data are expressed as the mean ± SEM of 5 independent experiments ( n = 5). The levels of protein (B) and mRNA (C) were determined by Western blot analysis and real-time PCR, respectively. mRNA results are expressed as fold changes compared to the DMSO group. β-ACTIN was used as internal reference protein. Data are expressed as the mean ± SEM of 3–5 independent experiments ( n = 3–4, B; n = 5, C). # P < 0.05, ## P < 0.01 versus DMSO-treated control group, * P < 0.05, ** P < 0.01 versus LPS + ATP-treated group. (D) The NLRP3-ASC interaction in THP-1 monocytes were measured by immuno-fluorescence assay. The nuclei were stained with DAPI in blue, and targeted protein was stained in green or red (magnification: 630 × ×). (E) ROS levels were detected using an ROS fluorescent probe in THP-1 monocytes (magnification: 400 × ). (F and G) Intracellular potassium (F) and ionic calcium (G) levels in the cell lysates of THP-1 monocytes. Data are expressed as the mean ± SEM of 5 independent experiments ( n = 5). # P < 0.05, ## P < 0.01 versus DMSO-treated control group, * P < 0.05, ** P < 0.01 versus LPS + ATP-treated group. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: TaqMan probes, including those for human-specific NLRP3 (Hs00918082), NF κ B1 (Hs00765730), IL-1 β (Hs00174097), IL-6 (Hs00174131 ) , ICAM-1 (Hs00164932), COX-2 (Hs00153133), TNF- α (Hs00174128), and GAPDH (Hs02786624); and mouse-specific Nlrp3 (Mm00840904), Nf κ b1 (Mm00476361), Il-1 β (Mm00434228), Il-6 (Mm00446190 ) , Icam-1 (Mm00516023), Cox-2 (Mm00478374), Tnf- α (Mm00443258), Cyp7a1 (Mm00484150), Cyp8b1 (Mm00501637), Cyp27a1 (Mm00470430), Cyp2b10 (Mm01972453), Cyp3a11 (Mm00731567), Ugt1a1 (Mm02603337), Abcb11 (Mm00445168), Abcc2 (Mm00496899), Abcc3 (Mm00551550), Abcc4 (Mm01226381), Slc01b2 (Mm00451510), Slc10a1 (Mm00441421), and Gapdh (Mm99999915), were acquired from Invitrogen (Carlsbad, CA, USA).

Techniques: Activation Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Western Blot, Real-time Polymerase Chain Reaction, Control, Fluorescence, Staining

Journal: Redox Biology

Article Title: Inhibition of NLRP3-mediated crosstalk between hepatocytes and liver macrophages by geniposidic acid alleviates cholestatic liver inflammatory injury

doi: 10.1016/j.redox.2022.102404

Figure Lengend Snippet: GPA protects against ANIT-induced acute liver inflammation by inhibiting NLRP3 inflammasome activation. C57BL/6 mice were orally administered with the vehicle (0.5% carboxymethylcellulose) as control group, UDCA (75 mg/kg) as positive control, GPA-H (100 mg/kg), GPA-M (50 mg/kg), or GPA-L (25 mg/kg) daily for seven days. On day 5, they were orally administered with ANIT (100 mg/kg). (A) Representative FACS image of mouse liver sample. After sacrificing the animals, liver macrophage were isolated, and the inflammatory infiltration was evaluated by FACS (n = 5). Data are expressed as the mean ± SEM of 5 independent mice liver samples ( n = 5). # P < 0.05, ## P < 0.01 versus vehicle-treated control group, * P < 0.05, ** P < 0.01 versus ANIT-treated group. (B) Hepatic F4/80 and CD68 protein expression measured by immunofluorescence assay. The nuclei were stained with DAPI in blue, and targeted protein was stained in green (F4/80) or red (CD68) (magnification: 630 × ). (C) Levels of pro-inflammatory cytokines in mouse liver, including Il-1β, Il-6, and Tnf-α were detected by ELISA. Data are expressed as the mean ± SEM of 5–6 independent mouse samples ( n = 5–6). # P < 0.05, ## P < 0.01 versus vehicle-treated control group, * P < 0.05, ** P < 0.01 versus ANIT-treated group. (D) Changes in spleen weight index (ratio of spleen weight to body weight) in mice with ANIT-induced cholestasis. Data are expressed as the mean ± SEM of 5 independent mouse samples ( n = 5). (E) mRNAs levels of NLRP3 and other proinflammatory genes in mouse liver were analyzed by real-time PCR. Data are expressed as the mean ± SEM of 5 independent mouse samples ( n = 5). Results are expressed as fold changes compared to the vehicle-treated control group. # P < 0.05, ## P < 0.01 versus vehicle-treated control group, * P < 0.05, ** P < 0.01 versus ANIT-treated group. (F) Levels of NLRP3 and NF-κB signaling proteins in liver lysates were analyzed by western blotting. β-ACTIN was used as internal reference protein. Data are expressed as the mean ± SEM of 3–4 independent mouse samples ( n = 3–4). # P < 0.05, ## P < 0.01 versus vehicle-treated control group, * P < 0.05, ** P < 0.01 versus ANIT-treated group. (G) ANIT-induced hepatic ROS levels were decreased after GPA treatment ( n = 3). # P < 0.05, ## P < 0.01 versus vehicle-treated control group, * P < 0.05, ** P < 0.01 versus ANIT-treated group. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: TaqMan probes, including those for human-specific NLRP3 (Hs00918082), NF κ B1 (Hs00765730), IL-1 β (Hs00174097), IL-6 (Hs00174131 ) , ICAM-1 (Hs00164932), COX-2 (Hs00153133), TNF- α (Hs00174128), and GAPDH (Hs02786624); and mouse-specific Nlrp3 (Mm00840904), Nf κ b1 (Mm00476361), Il-1 β (Mm00434228), Il-6 (Mm00446190 ) , Icam-1 (Mm00516023), Cox-2 (Mm00478374), Tnf- α (Mm00443258), Cyp7a1 (Mm00484150), Cyp8b1 (Mm00501637), Cyp27a1 (Mm00470430), Cyp2b10 (Mm01972453), Cyp3a11 (Mm00731567), Ugt1a1 (Mm02603337), Abcb11 (Mm00445168), Abcc2 (Mm00496899), Abcc3 (Mm00551550), Abcc4 (Mm01226381), Slc01b2 (Mm00451510), Slc10a1 (Mm00441421), and Gapdh (Mm99999915), were acquired from Invitrogen (Carlsbad, CA, USA).

Techniques: Activation Assay, Control, Positive Control, Isolation, Expressing, Immunofluorescence, Staining, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Western Blot

Journal: Redox Biology

Article Title: Inhibition of NLRP3-mediated crosstalk between hepatocytes and liver macrophages by geniposidic acid alleviates cholestatic liver inflammatory injury

doi: 10.1016/j.redox.2022.102404

Figure Lengend Snippet: GPA reduces BA-induced cellular inflammation by blocking the activation of NLRP3 inflammasome in hepatocytes and macrophages. (A) Wild-type primary (WT) mouse hepatocytes (PMHs) were pretreated with GPA for 12 h and treated with 200 μM TCA for 24 h. Levels of NLRP3 and proinflammatory mRNAs were determined by real-time PCR. Results are expression as fold changes compared to the DMSO group. Data are expressed as the mean ± SEM of 5 independent experiments ( n = 5). (B) Levels of Il-1β and Il-6 in cell-culture supernatants were quantified by ELISA. Data are expressed as the mean ± SEM of 5 independent experiments ( n = 5). # P < 0.05, ## P < 0.01 versus DMSO-treated control group, * P < 0.05, ** P < 0.01 versus TCA-treated group. Primary mouse hepatocytes with Nlrp3 -knockout ( Nlrp3 −/− PMHs) were pretreated with GPA for 12 h and treated with 200 μM TCA for 24 h. Levels of Nfκb1, Il-1β , and Tnf-α mRNA (C) and Il-1β and Tnf-α in cell-culture supernatants (D) were determined by real-time PCR and ELISA, respectively. mRNA results are expression as fold changes compared to the DMSO group. Data are expressed as the mean ± SEM of 5 independent experiments ( n = 5). # P < 0.05, ## P < 0.01 versus DMSO-treated control group, * P < 0.05, ** P < 0.01 versus TCA-treated group. PMHs and BMDM were isolated from WT and Nlrp3 −/− mice and cultivated on a conditioned medium (CM)-associated culture. (E – H) WT BMDM were exposed for 24 h to the CM derived from WT-PMHs(E and F) and Nlrp3 −/− PMHs(G and H) that were pretreated with or without GPA for 24 h and TCA for another 24 h. Levels of NLRP3 and proinflammatory mRNAs (E and G) and IL-1β (F and H) in cell-culture supernatants were determined by real-time PCR and ELISA, respectively. mRNA results are expressed as fold changes compared to the DMSO group. Data are expressed as the mean ± SEM of 5 independent experiments ( n = 5). # P < 0.05, ## P < 0.01 versus DMSO-treated control group, * P < 0.05, ** P < 0.01 versus TCA-treated group. (I – J) BMDM were pretreated with GPA for 12 h and treated with 200 μM TCA for 24 h. Levels of NLRP3 and proinflammatory mRNAs and pro-inflammatory cytokines in cell-culture supernatants were quantified by real-time PCR (I) and ELISA analysis (J), respectively. mRNA results are expressed as fold changes compared to the DMSO group. Data are expressed as the mean ± SEM of 5 independent experiments ( n = 5). # P < 0.05, ## P < 0.01 versus DMSO-treated control group, * P < 0.05, ** P < 0.01 versus TCA-treated group. (K–N) WT PMHs were exposed for 24 h to the CM derived from WT-BMDM (K and L) and Nlrp3 −/− (M and N) BMDM that were pretreated with or without GPA for 24 h, followed by TCA for 24 h. Levels of NLRP3 and proinflammatory mRNAs (K and M) and IL-1β (L and N) in cell-culture supernatants were determined by real-time PCR and ELISA analysis, respectively. mRNA results are expressed as fold changes compared to the DMSO group. Data are expressed as the mean ± SEM of 5 independent experiments ( n = 5). # P < 0.05, ## P < 0.01 versus DMSO-treated control group, * P < 0.05, ** P < 0.01 versus TCA-treated group.

Article Snippet: TaqMan probes, including those for human-specific NLRP3 (Hs00918082), NF κ B1 (Hs00765730), IL-1 β (Hs00174097), IL-6 (Hs00174131 ) , ICAM-1 (Hs00164932), COX-2 (Hs00153133), TNF- α (Hs00174128), and GAPDH (Hs02786624); and mouse-specific Nlrp3 (Mm00840904), Nf κ b1 (Mm00476361), Il-1 β (Mm00434228), Il-6 (Mm00446190 ) , Icam-1 (Mm00516023), Cox-2 (Mm00478374), Tnf- α (Mm00443258), Cyp7a1 (Mm00484150), Cyp8b1 (Mm00501637), Cyp27a1 (Mm00470430), Cyp2b10 (Mm01972453), Cyp3a11 (Mm00731567), Ugt1a1 (Mm02603337), Abcb11 (Mm00445168), Abcc2 (Mm00496899), Abcc3 (Mm00551550), Abcc4 (Mm01226381), Slc01b2 (Mm00451510), Slc10a1 (Mm00441421), and Gapdh (Mm99999915), were acquired from Invitrogen (Carlsbad, CA, USA).

Techniques: Blocking Assay, Activation Assay, Real-time Polymerase Chain Reaction, Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay, Control, Knock-Out, Isolation, Derivative Assay

Journal: Redox Biology

Article Title: Inhibition of NLRP3-mediated crosstalk between hepatocytes and liver macrophages by geniposidic acid alleviates cholestatic liver inflammatory injury

doi: 10.1016/j.redox.2022.102404

Figure Lengend Snippet: Anti-inflammatory effect of GPA against ANIT‐induced acute liver injury is largely abolished in Nlrp3 -knockout mice. WT mice and Nlrp3 −/− mice were orally administered with the vehicle (0.5% carboxymethylcellulose), UDCA (75 mg/kg) as positive control, or GPA-H (100 mg/kg) once daily for seven days and with ANIT (100 mg/kg) on day 5. (A) Representative FACS image of mouse liver sample. After sacrificing the animals, liver macrophage were isolated, and the inflammatory infiltration was evaluated by FACS (n = 5). Data are expressed as the mean ± SEM of 5 independent mice liver samples ( n = 5). # P < 0.05, ## P < 0.01 versus vehicle-treated control group in WT and Nlrp3 −/− mice; * P < 0.05, ** P < 0.01 compared to ANIT-treated alone group. (B) Hepatic F4/80 and CD68 protein expression measured by immunofluorescence assay. The nuclei were stained with DAPI in blue, and targeted protein was stained in red (F4/80) or green (CD68) (magnification: 630 × ).Data are expressed as the mean ± SEM of 3 independent mice liver samples ( n = 3). # P < 0.05, ## P < 0.01 versus vehicle-treated control group in WT and Nlrp3 −/− mice; * P < 0.05, ** P < 0.01 compared to ANIT-treated alone group. (C) Levels of NLRP3 and proinflammatory mRNAs in mouse liver were determined by real-time PCR. Data are expressed as the mean ± SEM of 5 independent mouse samples ( n = 5). Results are expressed as fold changes compared to the vehicle-treated control group. # P < 0.05, ## P < 0.01 versus vehicle-treated control group in WT and Nlrp3 −/− mice; * P < 0.05, ** P < 0.01 compared to ANIT-treated alone group. (D) Levels of inflammatory cytokines including Il-1β, Il-6, and Tnf-α in mouse liver were detected by ELISA. Data are expressed as the mean ± SEM of 5 independent mouse samples ( n = 5). # P < 0.05, ## P < 0.01 versus vehicle-treated control group, * P < 0.05, ** P < 0.01 versus ANIT-treated group. (E) Effects of GPA treatment on spleen weight index (ratio of spleen weight to body weight) of ANIT-induced mice. Data are expressed as the mean ± SEM of 5 independent mouse samples ( n = 5). Results are expressed as fold changes compared to the vehicle-treated control group. # P < 0.05, ## P < 0.01 versus vehicle-treated control group in WT and Nlrp3 −/− mice; * P < 0.05, ** P < 0.01 compared to ANIT-treated alone group. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: TaqMan probes, including those for human-specific NLRP3 (Hs00918082), NF κ B1 (Hs00765730), IL-1 β (Hs00174097), IL-6 (Hs00174131 ) , ICAM-1 (Hs00164932), COX-2 (Hs00153133), TNF- α (Hs00174128), and GAPDH (Hs02786624); and mouse-specific Nlrp3 (Mm00840904), Nf κ b1 (Mm00476361), Il-1 β (Mm00434228), Il-6 (Mm00446190 ) , Icam-1 (Mm00516023), Cox-2 (Mm00478374), Tnf- α (Mm00443258), Cyp7a1 (Mm00484150), Cyp8b1 (Mm00501637), Cyp27a1 (Mm00470430), Cyp2b10 (Mm01972453), Cyp3a11 (Mm00731567), Ugt1a1 (Mm02603337), Abcb11 (Mm00445168), Abcc2 (Mm00496899), Abcc3 (Mm00551550), Abcc4 (Mm01226381), Slc01b2 (Mm00451510), Slc10a1 (Mm00441421), and Gapdh (Mm99999915), were acquired from Invitrogen (Carlsbad, CA, USA).

Techniques: Knock-Out, Positive Control, Isolation, Control, Expressing, Immunofluorescence, Staining, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: Redox Biology

Article Title: Inhibition of NLRP3-mediated crosstalk between hepatocytes and liver macrophages by geniposidic acid alleviates cholestatic liver inflammatory injury

doi: 10.1016/j.redox.2022.102404

Figure Lengend Snippet: Hepatoprotective effect of GPA against ANIT‐induced acute liver injury is partly mitigated in Nlrp3 -knockout mice. (A) Serum biochemical indexes of cholestatic liver injury including ALT, AST, TBA and TBIL levels were evaluated. Data are expressed as the mean ± SEM from 5 to 6 independent mice samples (n = 5–6). # P < 0.05, ## P < 0.01 versus vehicle-treated control group in WT and Nlrp3 −/− mice; * P < 0.05, ** P < 0.01 compared to ANIT-treated alone group. (B) Representative images of haematoxylin and eosin staining in liver sections (magnification:200 × ). The circle indicated bleeding, inflammatory infiltration and hepatic necrosis.Data are expressed as the mean ± SEM of 5 independent mouse samples ( n = 5). Results are expressed as fold changes compared to the vehicle-treated control group. # P < 0.05, ## P < 0.01 versus vehicle-treated control group in WT and Nlrp3 −/− mice; * P < 0.05, ** P < 0.01 compared to ANIT-treated alone group. (C) Relative mRNA levels of BA synthesis- and transport-related genes in mouse liver were tested by real-time PCR. Data are expressed as the mean ± SEM of 5 independent mouse samples ( n = 5). Results are expressed as fold changes compared to the vehicle-treated control group. # P < 0.05, ## P < 0.01 versus vehicle-treated control group in WT and Nlrp3 −/− mice; * P < 0.05, ** P < 0.01 compared to ANIT-treated alone group.

Article Snippet: TaqMan probes, including those for human-specific NLRP3 (Hs00918082), NF κ B1 (Hs00765730), IL-1 β (Hs00174097), IL-6 (Hs00174131 ) , ICAM-1 (Hs00164932), COX-2 (Hs00153133), TNF- α (Hs00174128), and GAPDH (Hs02786624); and mouse-specific Nlrp3 (Mm00840904), Nf κ b1 (Mm00476361), Il-1 β (Mm00434228), Il-6 (Mm00446190 ) , Icam-1 (Mm00516023), Cox-2 (Mm00478374), Tnf- α (Mm00443258), Cyp7a1 (Mm00484150), Cyp8b1 (Mm00501637), Cyp27a1 (Mm00470430), Cyp2b10 (Mm01972453), Cyp3a11 (Mm00731567), Ugt1a1 (Mm02603337), Abcb11 (Mm00445168), Abcc2 (Mm00496899), Abcc3 (Mm00551550), Abcc4 (Mm01226381), Slc01b2 (Mm00451510), Slc10a1 (Mm00441421), and Gapdh (Mm99999915), were acquired from Invitrogen (Carlsbad, CA, USA).

Techniques: Knock-Out, Control, Staining, Real-time Polymerase Chain Reaction

Journal: Redox Biology

Article Title: Inhibition of NLRP3-mediated crosstalk between hepatocytes and liver macrophages by geniposidic acid alleviates cholestatic liver inflammatory injury

doi: 10.1016/j.redox.2022.102404

Figure Lengend Snippet: Liver-specific overexpression of NLRP3 antagonizes the hepatoprotective effect of GPA against ANIT-induced acute inflammatory liver injury. WT mice were intravenously injected with Adv-Con or Adv-Nlrp3 adenovirus (1 × 10 9 pf) via the tail vein for three days and then orally administered with MCC950 (20 mg/kg) or GPA (100 mg/kg). On day 8, they were orally administered with ANIT(100 mg/kg) to induce acute liver injury. (A) Protein levels of NLRP3 in the liver tissues. Actin was used as internal reference protein. Data are expressed as the mean ± SEM of 3 independent mouse samples ( n = 3). (B) Serum Il-1β and Tnf-α levels were detected by ELISA. Data are expressed as the mean ± SEM of 5 independent mouse samples ( n = 5). # P < 0.05, ## P < 0.01 versus vehicle-treated control group in WT and Nlrp3 oe mice; * P < 0.05, ** P < 0.01 compared to ANIT-treated alone group. (C) Serum biochemical indexes of cholestatic liver injury including ALT, AST, TBA and TBIL levels were evaluated. Data are expressed as the mean ± SE from 5 to 6 independent mice samples (n = 5–6). # P < 0.05, ## P < 0.01 versus vehicle-treated control group in WT and Nlrp3 oe mice; * P < 0.05, ** P < 0.01 compared to ANIT-treated alone group. (D) Representative images of haematoxylin and eosin staining in liver sections (magnification:200 × ). The circle indicated bleeding, inflammatory infiltration and hepatic necrosis. Data are expressed as the mean ± SE from 5 independent mice samples (n = 5). # P < 0.05, ## P < 0.01 versus vehicle-treated control group in WT and Nlrp3 oe mice; * P < 0.05, ** P < 0.01 compared to ANIT-treated alone group.

Article Snippet: TaqMan probes, including those for human-specific NLRP3 (Hs00918082), NF κ B1 (Hs00765730), IL-1 β (Hs00174097), IL-6 (Hs00174131 ) , ICAM-1 (Hs00164932), COX-2 (Hs00153133), TNF- α (Hs00174128), and GAPDH (Hs02786624); and mouse-specific Nlrp3 (Mm00840904), Nf κ b1 (Mm00476361), Il-1 β (Mm00434228), Il-6 (Mm00446190 ) , Icam-1 (Mm00516023), Cox-2 (Mm00478374), Tnf- α (Mm00443258), Cyp7a1 (Mm00484150), Cyp8b1 (Mm00501637), Cyp27a1 (Mm00470430), Cyp2b10 (Mm01972453), Cyp3a11 (Mm00731567), Ugt1a1 (Mm02603337), Abcb11 (Mm00445168), Abcc2 (Mm00496899), Abcc3 (Mm00551550), Abcc4 (Mm01226381), Slc01b2 (Mm00451510), Slc10a1 (Mm00441421), and Gapdh (Mm99999915), were acquired from Invitrogen (Carlsbad, CA, USA).

Techniques: Over Expression, Injection, Enzyme-linked Immunosorbent Assay, Control, Staining

Journal: PLoS Biology

Article Title: Short-chain fatty acids bind to apoptosis-associated speck-like protein to activate inflammasome complex to prevent Salmonella infection

doi: 10.1371/journal.pbio.3000813

Figure Lengend Snippet: (A) SCFAs promote the binding between ASC and NLRP3 in vitro. GST or GST-ASC was incubated with fluorescently labeled NLRP3 in the presence or absence of propionate (left), butyrate (middle), or lactate (right). Proteins were visualized by fluorescence imaging (top) or Coomassie Brilliant Blue staining (bottom). (B) Expression vector for FLAG-ASC and/or NLRP3 was transfected into HEK293T cells treated with propionate, butyrate, or lactate. Cell lysates were subjected to coimmunoprecipitation using anti-FLAG resin, and bound proteins were visualized by western blotting using anti-FLAG or anti-NLRP3 antibody. (C) U937 cells were incubated with LPS/ATP and treated with propionate (Pro), butyrate (But), or lactate (Lac). The IL-1β (left) and IL-18 (right) levels in the culture media were measured by ELISA. Data are the mean ± SD of three independent assays. * P < 0.05, ** P < 0.01. Data are listed in . (D) The effects of SCFA derivatives on IL-1β production were analyzed as shown above. C2, acetate; C3, propionate; C4, butyrate; C5, valerate; C6, caproate; C7, enanthate; C8, caprylate; Lac, lactate; MeP, 2-methylpropanoic acid; Ala, alanine; Mal, maleate; Suc, succinate; Cit, citrate. Data are the mean ± SD of three independent assays. * P < 0.05, ** P < 0.01 versus LPS/ATP treatment. Data are listed in . ASC, apoptosis-associated speck-like protein; GST, glutathione-S-transferase; IL, interleukin; IP, immunoprecipitation; LPS, lipopolysaccharide; NLRP, nucleotide-binding oligomerization domain-like receptor protein; SCFA, short-chain fatty acid; SD, standard deviation.

Article Snippet: Human NLRP3 cDNA (OriGene, Rockville, MD, USA, cat. no. SC110747) was amplified using the primers 5′-TTTGGATCCATGAAGATGGCAAGCACCCGC-3′ (forward) and 5′-TTTCTCGAGCTACCAAGAAGGCTCAAAGAC-3′ (reverse) and was introduced into the mammalian expression vector pCDNA3.1 (Thermo Fisher Scientific, cat. no. V79020).

Techniques: Binding Assay, In Vitro, Incubation, Labeling, Fluorescence, Imaging, Staining, Expressing, Plasmid Preparation, Transfection, Western Blot, Enzyme-linked Immunosorbent Assay, Immunoprecipitation, Standard Deviation

Journal: Journal of Innate Immunity

Article Title: Siglec-14 Enhances NLRP3-Inflammasome Activation in Macrophages

doi: 10.1159/000504323

Figure Lengend Snippet: Expression of Siglec-14 on macrophages increases IL-1β secretion through NLRP3 inflammasome activation. a THP1-EV, THP1-Sig14 and THP1-Sig5 cells were treated with 25 nM of phorbol-12-myristate-13-acetate for 48 h and stimulated with 5 mM of ATP or 10 μM of nigericin for 30 min then incubated for additional 2 h; secreted IL-1β in supernatants was determined by ELISA. b The amount of secreted mature IL-1β in the supernatants of a were determined using IL-1β sensor cells (Invivogen) and optical density (OD) measurements at 655 nm. c Cells were treated with caspase-1 inhibitor Ac-YVAD-CMK (YVAD), NLRP3 inhibitor CRID3 sodium salt (CRID3) or mock control for 1 h and stimulated with ATP or nigericin for an additional 2 h followed by ELISA to determine the secreted IL-1β level in supernatants. d Cells were treated with PBS, K+ buffer or Na+ buffer for 2 h and exposed to nigericin or buffer control for an additional 2 h. Culture supernatants were used to determine secreted IL-1β levels by ELISA. Error bars represent the SD of data means from 3 independent experiments. Significance of p values was classified as follows: * p < 0.05, ** p< 0.005, *** p < 0.001. EV, empty vector; IL-1β, interleukin-1β; unstim., unstimulated; inhib, inhibition.

Article Snippet: Immunoblot analyses were performed with antibodies recognizing IL-1β and NLRP3 (12703 and 13158, Cell Signaling, Danvers, MA, USA), ASC (676502, Biolegend, San Diego, CA, USA), pro-caspase-1 + p10 (ab179515, Abcam, Cambridge, MA, USA), and β-actin (GTX109639, GeneTex, Irvine, CA, USA).

Techniques: Expressing, Activation Assay, Incubation, Enzyme-linked Immunosorbent Assay, Control, Plasmid Preparation, Inhibition

Journal: Journal of Innate Immunity

Article Title: Siglec-14 Enhances NLRP3-Inflammasome Activation in Macrophages

doi: 10.1159/000504323

Figure Lengend Snippet: Siglec-14 boosts the macrophage NLRP3 inflammasome response to GBS.a THP1-EV, THP1-Sig14 and THP1-Sig5 cells were treated with GBS WT and ΔcylE mutant at MOI = 10 bacteria/cell and incubated for 16 h; IL-1β and IL-6 release in supernatants were measured by ELISA. b THP1-EV, THP1-Sig14, and THP1-Sig5 cells were treated with the non-β-protein-expressing GBS ΔcylE mutant A9O9 for 1 h, then exposed to ATP or buffer control for 30 min. IL-1β release in supernatants was assessed by ELISA. LDH release was also assessed. c Western immunoblot analysis of NLRP3, pro-caspase-1, pro-IL-1β, and β-actin (loading control) expression in THP1-EV, THP1‑Sig14 and THP1-Sig5 cells. d Caspase-1 activity in supernatants of GBS-infected THP1-EV, THP1-Sig14, and THP1-Sig5 cells was assessed by colorimetric assay and optical density (OD) measurements at 400 nm. e THP1-EV, THP1-Sig14, and THP1-Sig5 cells were treated with SPNWTand sialidase deficient ΔNanA mutant at MOI = 10 bacteria/cell and incubated for 16 h. IL-1β and IL-6 release in the supernatant was assessed. Error bars represent the SD of data means from 3 independent experiments. ** p < 0.005, *** p < 0.001. IL-1β, interleukin-1β; GBS, group B Streptococcus; WT, wild-type; LDH, lactate dehydrogenase; EV, empty vector; NLRP3, nucleotide-binding domain leucine-rich repeat protein 3; unstim., unstimulated; SPN, Streptococcus pneumoniae

Article Snippet: Immunoblot analyses were performed with antibodies recognizing IL-1β and NLRP3 (12703 and 13158, Cell Signaling, Danvers, MA, USA), ASC (676502, Biolegend, San Diego, CA, USA), pro-caspase-1 + p10 (ab179515, Abcam, Cambridge, MA, USA), and β-actin (GTX109639, GeneTex, Irvine, CA, USA).

Techniques: Mutagenesis, Bacteria, Incubation, Enzyme-linked Immunosorbent Assay, Expressing, Control, Western Blot, Activity Assay, Infection, Colorimetric Assay, Plasmid Preparation, Binding Assay

Journal: Journal of Innate Immunity

Article Title: Siglec-14 Enhances NLRP3-Inflammasome Activation in Macrophages

doi: 10.1159/000504323

Figure Lengend Snippet: Vimentin can engage Siglec-14 to increase inflammasome activation. a Cell lysates of THP1-EV, THP1-Sig14 and THP1-Sig5 cells were immunoprecipitated with anti-Siglec-5/14 antibody and the interaction of vimentin assessed by western blotting. b Direct binding of Siglec5-Fc (Sig5), Siglec7-Fc (Sig7), Siglec9-Fc (Sig9) and Siglec14-Fc (Sig14) to recombinant vimentin as measured by ELISA; GBS β-protein immunoglobulin A binding region (IgA BR) was used as a negative control. c Direct binding of Siglec-14 and −5 to the various concentrations of recombinant vimentin measured by ELISA. d Cells were treated with 10 μg/mL of recombinant vimentin for the indicated time points, then phosphorylation of p38 mitogen-activated protein kinase P38 (p-P38) and AKT (p-AKT) protein levels were assessed by western blotting. e Cells were treated +/– recombinant vimentin for 2 h, then exposed to nigericin or buffer control for 30 min. Released IL-1β was assessed by ELISA. f Cells were treated with or without recombinant vimentin +/– NLRP3 inhibitor CRID3 sodium salt (CRID3) for 1 h, then exposed to nigericin or buffer control for an additional 2 h. IL-1β release was assessed by ELISA. Error bars represent the SD of data means from 3 independent experiments. * p < 0.05. IL-1β, interleukin-1β; EV, empty vector.

Article Snippet: Immunoblot analyses were performed with antibodies recognizing IL-1β and NLRP3 (12703 and 13158, Cell Signaling, Danvers, MA, USA), ASC (676502, Biolegend, San Diego, CA, USA), pro-caspase-1 + p10 (ab179515, Abcam, Cambridge, MA, USA), and β-actin (GTX109639, GeneTex, Irvine, CA, USA).

Techniques: Activation Assay, Immunoprecipitation, Western Blot, Binding Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Negative Control, Phospho-proteomics, Control, Plasmid Preparation

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Colchicine Ameliorates Dilated Cardiomyopathy Via SIRT2 ‐Mediated Suppression of NLRP3 Inflammasome Activation

doi: 10.1161/JAHA.122.025266

Figure Lengend Snippet: Primer Sequences for Real‐Time PCR Analysis

Article Snippet: To clarify the effect of colchicine on the inflammasome, primary antibodies were added to heart sections overnight at a dilution of 1:100 for apoptosis‐associated speck‐like protein with CARD domain (PA5‐95826, Invitrogen), 1:100 for NLRP3 (768319, Novus), and 1:100 for SIRT2 (19655‐1‐AP, Proteintech) at 4 °C.

Techniques: Real-time Polymerase Chain Reaction

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Colchicine Ameliorates Dilated Cardiomyopathy Via SIRT2 ‐Mediated Suppression of NLRP3 Inflammasome Activation

doi: 10.1161/JAHA.122.025266

Figure Lengend Snippet: A and B , Representative immunoblots and the corresponding analysis of NLRP3 inflammasome pathway in cardiac tissues at the end point of experiment. GAPDH shows as loading control. C , Representative immunofluorescence images of cardiac tissues staining with ASC (green), cTNT (cardiac troponin T) (red), and DAPI (blue). Frequency of ASC speck is shown on the right (n=6–8 each group). Five images per mouse are evaluated. Scale bar=50 μm. Data are shown as mean±SEM. All data statistical significance are determined by 1‐way ANOVA (Tukey post‐test). cTNT indicates cardiac troponin T; DCM, dilated cardiomyopathy; IL‐1β, interleukin‐1β; and NLRP3, NOD‐like receptor protein 3. * P <0.05 between groups; ** P <0.01 between groups; *** P <0.005 between groups; and **** P <0.001 between groups. AIM2 indicates absent in melanoma 2; ASC, apoptosis‐associated speck‐like protein with CARD domain; Col, colchicine.

Article Snippet: To clarify the effect of colchicine on the inflammasome, primary antibodies were added to heart sections overnight at a dilution of 1:100 for apoptosis‐associated speck‐like protein with CARD domain (PA5‐95826, Invitrogen), 1:100 for NLRP3 (768319, Novus), and 1:100 for SIRT2 (19655‐1‐AP, Proteintech) at 4 °C.

Techniques: Western Blot, Immunofluorescence, Staining

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Colchicine Ameliorates Dilated Cardiomyopathy Via SIRT2 ‐Mediated Suppression of NLRP3 Inflammasome Activation

doi: 10.1161/JAHA.122.025266

Figure Lengend Snippet: A , Acetylated NLRP3 (NOD‐like receptor protein 3) levels in the cardiac tissues of mice at the end point of experiment. Immunoglobulin G is the negative control and GAPDH is the input loading control. Data are representative of 3 independent experiments. Western blot analysis on expression of SIRT2 in cardiac tissues at the end of experiment (n=6 each group) on the right. B , Relative expression of SIRT2 mRNA is normalized to GAPDH mRNA in cardiac tissues (n=4 each group). C , Relative expression of inflammatory cytokine interleukin‐1 mRNA and NLRP3 mRNA are normalized to GAPDH mRNA in primary neutrophil that are treated +/− colchicine (10 μmol/L, 2 hours), +/− SIRT2 inhibitor AGK2 (10 μmol/L, 2 hours) or challenged with doxorubicin (5 μmol/L, 2 hours) and inflammasome activators nigericin (10 μmol/L, 2 hours); n=6 each group. D , Western blot analysis on expression of NLRP3 inflammasome pathway in primary neutrophil that are treated +/− colchicine (10 μmol/L, 2 hours), +/− SIRT2 inhibitor AGK2 (10 μmol/L, 2 hours) or challenged with doxorubicin (5 μmol/L, 2 hours) and inflammasome activators nigericin (10 μmol/L, 2 hours). GAPDH shows loading control. Data are representative of 3 independent experiments. Data are shown as mean±SEM. Data of ( A and B ) are analyzed by 1‐way ANOVA (Tukey post‐test). Data of ( C and D ) are analyzed by 2‐way ANOVA (Bonferroni post‐test). DCM indicates dilated cardiomyopathy; IL‐1β, interleukin‐1β; NLRP3, NOD‐like receptor protein 3; and SIRT2, Sirtuin 2. * P <0.05 between groups; ** P <0.01 between groups; *** P <0.005 between groups; and **** P <0.001 between groups. AGK2 indicates a reversible inhibitor of SIRT2; AIM2, absent in melanoma 2; ASC, apoptosis‐associated speck‐like protein with CARD domain; Col, colchicine; IB, immunoblot; IP, immunoprecipitation; Ly6G, lymphocyte antigen 6 complex locus G6D.

Article Snippet: To clarify the effect of colchicine on the inflammasome, primary antibodies were added to heart sections overnight at a dilution of 1:100 for apoptosis‐associated speck‐like protein with CARD domain (PA5‐95826, Invitrogen), 1:100 for NLRP3 (768319, Novus), and 1:100 for SIRT2 (19655‐1‐AP, Proteintech) at 4 °C.

Techniques: Negative Control, Western Blot, Expressing, Immunoprecipitation

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Colchicine Ameliorates Dilated Cardiomyopathy Via SIRT2 ‐Mediated Suppression of NLRP3 Inflammasome Activation

doi: 10.1161/JAHA.122.025266

Figure Lengend Snippet: A , Representative immunofluorescence images of cardiac tissues staining with NLRP3 (green), SIRT2 (red), and DAPI (blue). (n=6–8 each group). Five images per mouse are evaluated. Scale bar=50 μm. B , Representative immunofluorescence images of primary neutrophils that are treated +/− colchicine (10 μmol/L, 2 hours), +/− SIRT2 inhibitor AGK2 (10 μmol/L, 2 hours) or challenged with doxorubicin (5 μmol/L, 2 hours) and inflammasome activators nigericin (10 μmol/L, 2 hours). NLRP3 (green), SIRT2 (red) and DAPI (blue). Scale bar=20 μm. Data are representative of 3 independent experiments. C , The effect of SIRT2 on the deacetylation of NLRP3 is evaluated by coimmunoprecipitation. NLRP3‐Flag plasmid is transfected into NB4 cell line, then NB4 cell line is treated with +/− colchicine (10 μmol/L, 2 hours), +/− SIRT2 inhibitor AGK2 (10 μmol/L, 2 hours) or challenged with doxorubicin (5 μmol/L, 2 hours) and inflammasome activators nigericin (10 μmol/L, 2 hours). Cell lysates are immunopurified with anti‐Flag antibody. Then the expression of acetylated lysine is measured by IP. GAPDH is the input loading control. Data are representative of 3 independent experiments. DCM indicates dilated cardiomyopathy; IL‐1β, interleukin‐1β; NLRP3, NOD‐like receptor protein 3; and SIRT2, Sirtuin 2. AGK2, a reversible inhibitor of SIRT2; Col, colchicine; IB, immunoblot; IP, Immunoprecipitation.

Article Snippet: To clarify the effect of colchicine on the inflammasome, primary antibodies were added to heart sections overnight at a dilution of 1:100 for apoptosis‐associated speck‐like protein with CARD domain (PA5‐95826, Invitrogen), 1:100 for NLRP3 (768319, Novus), and 1:100 for SIRT2 (19655‐1‐AP, Proteintech) at 4 °C.

Techniques: Immunofluorescence, Staining, Plasmid Preparation, Transfection, Expressing, Western Blot, Immunoprecipitation

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: α7 nicotinic acetylcholine receptor signaling inhibits inflammasome activation by preventing mitochondrial DNA release.

doi: 10.2119/molmed.2013.00117

Figure Lengend Snippet: Figure 2. α7 nAchR negatively regulates inflammasome activation. (A) LPS-primed peri- toneal mouse macrophages from either WT or α7 nAchR knockout (a7 KO) mice were stimulated with ATP in the presence or the absence of acetylcholine for 30 min. (B) WT and a7 KO mice were injected intraperitoneally with LPS (10 mg/kg). Serum samples were collected 2.5 h later. (C) HEK293A cells were transfected with plasmids as indicated. At 24 h after transfection, cell lysates were harvested. IL-1β cleavage and caspase-1 activa- tion were assessed by Western blot analysis. Serum levels of IL-1β, IL-18 or IL-6 were mea- sured by ELISA. Blots are representative of three independent experiments. Data shown are means ± SD.

Article Snippet: Plasmids expressing procaspase-1, ASC, NLRP3 or α7 nAchR were purchased from Origene (Rockville, MD, USA).

Techniques: Activation Assay, Knock-Out, Injection, Transfection, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: α7 nicotinic acetylcholine receptor signaling inhibits inflammasome activation by preventing mitochondrial DNA release.

doi: 10.2119/molmed.2013.00117

Figure Lengend Snippet: Figure 6. The mechanisms by which acetylcholine inhibits inflammasome activation. Extra- cellular ATP activates the NLRP3 inflammasome by provoking intracellular potassium efflux and mitochondrial DNA release into cytoplasm. Stimulation of ATP also results in rapid in- flux of acetylcholine into cytoplasm. There, acetylcholine prevents mitochondrial DNA re- lease through α7 nAchR and subsequently inhibits the NLRP3 inflammasome activation.

Article Snippet: Plasmids expressing procaspase-1, ASC, NLRP3 or α7 nAchR were purchased from Origene (Rockville, MD, USA).

Techniques: Activation Assay

Journal: Advanced Science

Article Title: Ciclopirox Olamine Inhibits the NLRP3 Inflammasome to Alleviate Inflammatory Diseases

doi: 10.1002/advs.75704

Figure Lengend Snippet: Identification of CPX as a potential inhibitor of NLRP3 Inflammasome. (A) Screening workflow of 190 FDA‐approved drugs. The THP‐1 cells (pretreated with 200 ng/mL PMA overnight) were primed with LPS, and incubated with drugs (10 µ m ) for 1 h prior to nigericin stimulation. Then, IL‐1β secretion was quantified by ELISA. (B) IL‐1β inhibition rate in culture supernatants (Sup) from THP‐1 cells. (C) The chemical structure of CPX. (D) IC 50 of CPX on the NLRP3 inflammasome‐dependent IL‐1β secretion (0–80 µ m ). (E,F) LPS‐primed THP‐1 cells were treated with various doses of CPX or DMSO for 1 h and then stimulated with nigericin. (E) ELISA of IL‐1β in Sup from THP‐1 cells. (F) Immunoblot analysis of matured‐IL‐1β and cleaved caspase‐1 in Sup, and NLRP3, pro‐IL‐1β, pro‐caspase‐1, and ASC in lysates (Lys) from THP‐1 cells. (G) The cytotoxicity of CPX on THP‐1 cells was measured using the CCK‐8 kit. Data from three independent experiments (E. Values are mean ± SD) or are representative of three independent experiments(F). Statistical analyses were carried out via one‐way ANOVA for (E), ** p < 0.01, *** p < 0.001.

Article Snippet: Ciclopirox olamine, imiquimod, TcdB toxin, and recombinant human NLRP3 were purchased from MedChemExpress (New Jersey, USA); Phorbol‐12‐myristate‐13‐acetate(PMA), LPS, nigericin, LDH activity assay kit, MitoTracker, MitoSOX, RIPA, human IgG, penicillin/streptomycin, NP40, and protein A+G agarose were supplied by Beyotime (Shanghai, China); MSU, SiO 2 , Alum, poly(dA:dT), pronase, disuccinimidyl suberate, and anti‐MYC were purchased from Sigma (St, Louis, USA); ATP, lipofectamine 2000, and Salmonella typhimurium (FLA‐ST) were supplied by InvivoGen (New Jersey, USA); and the ADP‐Glo Kinase assay kit was purchased from Promega (WI, USA).

Techniques: Incubation, Enzyme-linked Immunosorbent Assay, Inhibition, Western Blot, CCK-8 Assay

Journal: Advanced Science

Article Title: Ciclopirox Olamine Inhibits the NLRP3 Inflammasome to Alleviate Inflammatory Diseases

doi: 10.1002/advs.75704

Figure Lengend Snippet: CPX blocks NLRP3 inflammasome activation. (A,B) THP‐1 cells were first primed with LPS (3 h) and then treated with or without 20 µ m CPX for 1 h, followed by stimulation with ATP for 40 min, nigericin for 1 h, or MSU for 6 h. ELISA of IL‐1β in Sup (A), or immunoblot analysis of matured‐IL‐1β and cleaved caspase‐1 in Sup. (C,D) LPS‐primed BMDMs were treated with 20 µ m CPX and then stimulated with ATP for 40 min, nigericin for 1 h, or MSU for 6 h. ELISA of IL‐1β in Sup (C), or immunoblot analysis of matured‐IL‐1β and cleaved caspase‐1 in Sup (D). (E‐F) THP‐1 cells were primed with LPS (3 h) and then treated with or without 20 µ m CPX for 1 h, followed by stimulation with Alum for 6 h, imiquimod for 3 h, or SiO2 for 6 h. ELISA of IL‐1β in Sup (E), or immunoblot analysis of matured‐IL‐1β and cleaved caspase‐1 in Sup(F). (G,H) THP‐1 cells were treated and primed with LPS (3 h) and then treated with or without 20 µ m CPX for 1 h, followed by stimulation with poly(dA:dT) for 4 h, FLA‐ST for 4 h, or TcdB for 1 h. ELISA of IL‐1β in Sup (G) or immunoblot analysis of matured‐IL‐1β and cleaved caspase‐1 in Sup (H). (I) Representative microscopic images of LPS‐primed THP‐1 cells upon nigericin stimulation with or without CPX pre‐treatment (red arrows mark pyroptotic cells). Scale bars correspond to 100 µm. (J) PI (red fluorescence) and Hoechst (blue fluorescence) staining of THP‐1 cells revealed the dead cells. Scale bars correspond to 100 µm. (K) The release of LDH from LPS‐primed THP‐1 cells, pre‐treated with or without CPX and then stimulated with ATP, nigericin, or MSU. (L) Immunoblot analysis of GSDMD cleavage from THP‐1 cells, primed with LPS before treatment with or without CPX, followed by treatment with nigericin. Data from three independent experiments (A,C,E,G,K. Values are mean ± SD) or are representative of three independent experiments with similar results (B,D,F,H–J). Statistical analyses were carried out via two‐way ANOVA for (A,C,E,G,K), *** p < 0.001, NS not significant.

Article Snippet: Ciclopirox olamine, imiquimod, TcdB toxin, and recombinant human NLRP3 were purchased from MedChemExpress (New Jersey, USA); Phorbol‐12‐myristate‐13‐acetate(PMA), LPS, nigericin, LDH activity assay kit, MitoTracker, MitoSOX, RIPA, human IgG, penicillin/streptomycin, NP40, and protein A+G agarose were supplied by Beyotime (Shanghai, China); MSU, SiO 2 , Alum, poly(dA:dT), pronase, disuccinimidyl suberate, and anti‐MYC were purchased from Sigma (St, Louis, USA); ATP, lipofectamine 2000, and Salmonella typhimurium (FLA‐ST) were supplied by InvivoGen (New Jersey, USA); and the ADP‐Glo Kinase assay kit was purchased from Promega (WI, USA).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Fluorescence, Staining

Journal: Advanced Science

Article Title: Ciclopirox Olamine Inhibits the NLRP3 Inflammasome to Alleviate Inflammatory Diseases

doi: 10.1002/advs.75704

Figure Lengend Snippet: CPX inhibits NLRP3 inflammasome activation in vivo and in patient cells. (A–J) C57BL/6 mice were intraperitoneally injected with vehicle, CPX (10 mg/kg), or CPX (20 mg/kg) for 1 h prior to LPS injection to induce sepsis. (A) Schematic diagram of the LPS‐induced sepsis model. (B) Survival analysis of mice pretreated with vehicle or CPX (20 mg/kg) for 1 h before being intraperitoneally injected with LPS ( n = 10). (C,D) The activity of plasma ALT and AST ( n = 6). (E) Representative images of H&E‐stained liver sections. Scale bars, 50 µm. (F–I) ELISA of IL‐1β and IL‐6 in serum and in Sup from cultured liver tissues ( n = 6). (F) IL‐1β and (G) IL‐6 levels in serum. (H) IL‐1β and (I) IL‐6 levels in liver tissues. (J) Immunoblot analysis of matured‐IL‐1β and cleaved caspase‐1 in liver tissues. (K–R) C57BL/6 mice were treated with 3% DSS in drinking water for 6 days, followed by 4 days of normal drinking water. CPX or vehicle was administered daily ( n = 6). (K) Schematic diagram of DSS‐induced colitis model. (L) Body weights, (M) the disease activity index (DAI), (N) representative colon images, and (O) colon length were measured. (P) ELISA of IL‐1β in colon tissues. (Q) H&E‐stained colon tissues. (R) Immunoblot analysis of matured‐IL‐1β and cleaved caspase‐1 in colon tissues. (S,T) PBMCs primed with LPS and treated with 20 µ m CPX for 1 h, and then stimulated with nigericin or MSU. ELISA of IL‐1β in Sup of PBMCs isolated from healthy individuals (S) or gout patients (T). Date from three independent experiments (S,T. Values are mean ± SD). Statistical analyses were carried out via two‐way ANOVA for (B–D, F–L, L,M, O,P, S,T), * p < 0.05, ** p < 0.01, *** p < 0.001, NS not significant.

Article Snippet: Ciclopirox olamine, imiquimod, TcdB toxin, and recombinant human NLRP3 were purchased from MedChemExpress (New Jersey, USA); Phorbol‐12‐myristate‐13‐acetate(PMA), LPS, nigericin, LDH activity assay kit, MitoTracker, MitoSOX, RIPA, human IgG, penicillin/streptomycin, NP40, and protein A+G agarose were supplied by Beyotime (Shanghai, China); MSU, SiO 2 , Alum, poly(dA:dT), pronase, disuccinimidyl suberate, and anti‐MYC were purchased from Sigma (St, Louis, USA); ATP, lipofectamine 2000, and Salmonella typhimurium (FLA‐ST) were supplied by InvivoGen (New Jersey, USA); and the ADP‐Glo Kinase assay kit was purchased from Promega (WI, USA).

Techniques: Activation Assay, In Vivo, Injection, Activity Assay, Clinical Proteomics, Staining, Enzyme-linked Immunosorbent Assay, Cell Culture, Western Blot, Isolation

Journal: Advanced Science

Article Title: Ciclopirox Olamine Inhibits the NLRP3 Inflammasome to Alleviate Inflammatory Diseases

doi: 10.1002/advs.75704

Figure Lengend Snippet: CPX inhibits NLRP3 inflammasome assembly. (A) ELISA of TNF‐α in Sup from THP‐1 cells treated with various doses of CPX and then stimulated with 500 ng/mL LPS for 3 h. (B) ELISA of TNF‐α and (C) immunoblot analysis of NLRP3, pro‐caspase‐1, pro‐IL‐1β, and ASC in Lys from THP‐1 cells treated with 20 µ m CPX before or after stimulation with 500 ng/mL LPS for 3 h. (D,E) Qualification of potassium and chloride ions in LPS‐primed THP‐1 cells pretreated with or without 20 µ m CPX and then stimulated with nigericin. (F,G) Qualification of mtROS in THP‐1 cells was primed with LPS, pre‐treated with or without CPX, and subsequently stimulated with nigericin. (H) Immunoblot analysis of ASC oligomerization. (I) Representative images of ASC specks detected by immunofluorescence. DAPI shows nuclei, and white arrows show an example of ASC specks formed. Scale bar corresponds to 50 µm. (J) Percentage of ASC speck‐positive cells. (K) IP and immunoblot analysis to evaluate the NLRP3‐ASC interaction in LPS‐primed THP‐1 cells pretreated with CPX and then stimulated with nigericin. (L) IP and immunoblot analysis of HEK‐293T cells transfected with FLAG‐NLRP3, HIS‐ASC plasmids as indicated and treated with CPX. (M) IP and immunoblot analysis to evaluate the NLRP3‐NEK7 interaction in LPS‐primed THP‐1 cells pretreated with CPX and then stimulated with nigericin. (N) IP and immunoblot analysis of HEK‐293T cells transfected with FLAG‐NLRP3, MYC‐NEK7 plasmids as indicated, and treated with the CPX Data from three independent experiments(A,B,D,E,G,J. Values are mean ± SD) or are representative of three independent experiments with similar results(F,H,I,K–N). Statistical analyses were carried out via two‐way ANOVA for (A,B,D,E,G,J), *** p < 0.001, NS not significant.

Article Snippet: Ciclopirox olamine, imiquimod, TcdB toxin, and recombinant human NLRP3 were purchased from MedChemExpress (New Jersey, USA); Phorbol‐12‐myristate‐13‐acetate(PMA), LPS, nigericin, LDH activity assay kit, MitoTracker, MitoSOX, RIPA, human IgG, penicillin/streptomycin, NP40, and protein A+G agarose were supplied by Beyotime (Shanghai, China); MSU, SiO 2 , Alum, poly(dA:dT), pronase, disuccinimidyl suberate, and anti‐MYC were purchased from Sigma (St, Louis, USA); ATP, lipofectamine 2000, and Salmonella typhimurium (FLA‐ST) were supplied by InvivoGen (New Jersey, USA); and the ADP‐Glo Kinase assay kit was purchased from Promega (WI, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Immunofluorescence, Transfection

Journal: Advanced Science

Article Title: Ciclopirox Olamine Inhibits the NLRP3 Inflammasome to Alleviate Inflammatory Diseases

doi: 10.1002/advs.75704

Figure Lengend Snippet: CPX directly targets NLRP3 and inhibits its oligomerization. (A) The schematic shows the steps for identifying the target of CPX in THP‐1 cells using drug affinity responsive target stability (DARTS) and mass spectrometry analysis. (B) Coomassie blue staining of DARTS assay using THP‐1 cell lysates treated with or without CPX. (C) The target protein of CPX was identified by mass spectrogram. (D) DARTS in THP‐1 cells incubation with CPX or DMSO. (E) DARTS in HEK‐293T cells transfected with the FLAG‐NLRP3 plasmid under incubation with CPX or DMSO. (F) CESTA of NLRP3 in THP‐1 cells incubated with CPX or DMSO. (G) The melting curve generated from CETSA. (H) The oligomerization of NLRP3 in THP‐1 cells treated with CPX was analyzed by native‐PAGE. (I) NLRP3 oligomerization in HEK‐293T cells expressing FLAG‐NLRP3 plasmids and treated with CPX. (J) IP and immunoblot analysis of HEK‐293T cells transfected with FLAG‐NLRP3 and MYC‐NLRP3 plasmids as indicated and treated with CPX. (K) ELISA of IL‐1β in Sup and immunoblot analysis of matured‐IL‐1β and cleaved caspase‐1 in Sup from WT and NLRP3 −/− BMDMs were first primed with LPS and then treated with CPX, followed by stimulation with nigericin. (L) ELISA of IL‐1β in Sup and immunoblot analysis in Lys from HEK‐293T cells transfected with HA‐IL‐1β, HA‐pro‐caspase‐1, HIS‐ASC, with or without FLAG‐NLRP3 plasmids as indicated and treated with CPX. Data from three independent experiments (G,K,L. Values are mean ± SD) or are representative of three independent experiments with similar results (D,F,H–L). Statistical analyses were carried out via two‐way ANOVA for (K,L), ** p < 0.01, *** p < 0.001, NS not significant.

Article Snippet: Ciclopirox olamine, imiquimod, TcdB toxin, and recombinant human NLRP3 were purchased from MedChemExpress (New Jersey, USA); Phorbol‐12‐myristate‐13‐acetate(PMA), LPS, nigericin, LDH activity assay kit, MitoTracker, MitoSOX, RIPA, human IgG, penicillin/streptomycin, NP40, and protein A+G agarose were supplied by Beyotime (Shanghai, China); MSU, SiO 2 , Alum, poly(dA:dT), pronase, disuccinimidyl suberate, and anti‐MYC were purchased from Sigma (St, Louis, USA); ATP, lipofectamine 2000, and Salmonella typhimurium (FLA‐ST) were supplied by InvivoGen (New Jersey, USA); and the ADP‐Glo Kinase assay kit was purchased from Promega (WI, USA).

Techniques: Mass Spectrometry, Staining, Incubation, Transfection, Plasmid Preparation, Generated, Clear Native PAGE, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Advanced Science

Article Title: Ciclopirox Olamine Inhibits the NLRP3 Inflammasome to Alleviate Inflammatory Diseases

doi: 10.1002/advs.75704

Figure Lengend Snippet: CPX binds to tyrosine 381 of the NACHT domain. (A,B) LPS‐primed THP‐1 cells were treated with CPX for 30 min and washed out, then stimulated with nigericin. (A) ELISA of IL‐1β in Sup. (B) Immunoblot analysis of matured‐IL‐1β and cleaved caspase‐1 in Sup. (C) Schematic of the protein domains of NLRP3. (D–F) DARTS in HEK293T transfected with (D) FLAG‐NACHT domain, (E) FLAG‐PYD domain, and (F) FLAG‐LRR domain plasmids under incubation with CPX or DMSO. (G,H) The binding affinity of CPX to the NLRP3‐NACHT domain was measured by SPR assay. (I) Molecular docking analysis of CPX bound to NLRP3‐NACHT domain. CPX (yellow, stick model) in the binding site of NLRP3‐NACHT domain (lavender, cartoon, and PDB ID: 7ALV). (J) Structural comparison of the complex at five moments (0, 25, 50, 75, 100 ns) during a 100 ns molecular dynamics simulation. (K) Energy contribution of amino acid residues involved in binding. (L) DARTS in HEK293T transfected with R167A NACHT domain or Y381A NACHT domain plasmids under incubation with CPX or DMSO. (M) IP and immunoblot analysis of HEK293T cells transfected with MYC‐NEK7, FLAG‐NLRP3, or Y381A mutant FLAG‐NLRP3 plasmids and treated with 20 µ m CPX. (N) IP and immunoblot analysis of HEK293T cells transfected with HIS‐ASC, FLAG‐NLRP3, or Y381A mutant FLAG‐NLRP3 plasmids and treated with 20 µ m CPX. (O) NLRP3 oligomerization in HEK‐293T cells transfected with FLAG‐NLRP3 or Y381A mutant FLAG‐NLRP3 plasmids and treated with CPX. (R) LPS‐primed NLRP3‐/‐ BMDMs reconstituted with WT or Y381A mutant NLRP3, were treated with CPX and then stimulated with nigericin. ELISA of IL‐1β in Sup, immunoblot analysis of matured‐IL‐1β and cleaved caspase‐1 in Sup. (Q) ATPase activity assay for purified human NLRP3 in the presence of different concentrations of CPX. Data from three independent experiments (A, P, Q. Values are mean ± SD) or are representative of three independent experiments with similar results (B, D–F, L–P). Statistical analyses were carried out via two‐way ANOVA for (A, P, Q), ** p < 0.01, *** p < 0.001, NS not significant.

Article Snippet: Ciclopirox olamine, imiquimod, TcdB toxin, and recombinant human NLRP3 were purchased from MedChemExpress (New Jersey, USA); Phorbol‐12‐myristate‐13‐acetate(PMA), LPS, nigericin, LDH activity assay kit, MitoTracker, MitoSOX, RIPA, human IgG, penicillin/streptomycin, NP40, and protein A+G agarose were supplied by Beyotime (Shanghai, China); MSU, SiO 2 , Alum, poly(dA:dT), pronase, disuccinimidyl suberate, and anti‐MYC were purchased from Sigma (St, Louis, USA); ATP, lipofectamine 2000, and Salmonella typhimurium (FLA‐ST) were supplied by InvivoGen (New Jersey, USA); and the ADP‐Glo Kinase assay kit was purchased from Promega (WI, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Transfection, Incubation, Binding Assay, SPR Assay, Comparison, Mutagenesis, Activity Assay, Purification

Journal: Advanced Science

Article Title: Ciclopirox Olamine Inhibits the NLRP3 Inflammasome to Alleviate Inflammatory Diseases

doi: 10.1002/advs.75704

Figure Lengend Snippet: The mechanism of CPX blocking NLRP3 inflammasome activation.

Article Snippet: Ciclopirox olamine, imiquimod, TcdB toxin, and recombinant human NLRP3 were purchased from MedChemExpress (New Jersey, USA); Phorbol‐12‐myristate‐13‐acetate(PMA), LPS, nigericin, LDH activity assay kit, MitoTracker, MitoSOX, RIPA, human IgG, penicillin/streptomycin, NP40, and protein A+G agarose were supplied by Beyotime (Shanghai, China); MSU, SiO 2 , Alum, poly(dA:dT), pronase, disuccinimidyl suberate, and anti‐MYC were purchased from Sigma (St, Louis, USA); ATP, lipofectamine 2000, and Salmonella typhimurium (FLA‐ST) were supplied by InvivoGen (New Jersey, USA); and the ADP‐Glo Kinase assay kit was purchased from Promega (WI, USA).

Techniques: Blocking Assay, Activation Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: KIF1B Regulates NLRP3 ‐Mediated Pyroptosis in Asthma Progression

doi: 10.1111/jcmm.70975

Figure Lengend Snippet: KIF1B inhibition reduces IL‐13‐induced inflammatory responses in BEAS‐2B cells. (A) BEAS‐2B cells were transfected with si‐KIF1B or si‐NC and treated with IL‐13 (10 ng/mL) for 24 h, followed by Western blot analysis of NLRP3, cleaved caspase‐1 and cleaved GSDMD protein expression. Conditioned medium was collected to measure cytokine levels: (B) TNF‐α, (C) IL‐1β, (D) IL‐18 and (E) IL‐10 by ELISA. β‐actin was used as the loading control for normalisation in Western blot. Data are presented as mean ± SD ( n = 3; *** p < 0.001 vs. control, p < 0.01, p < 0.001 vs. IL‐13 group).

Article Snippet: Upon reaching approximately 60%–70% confluency, cells were transfected with si‐KIF1B (1:1000 dilution, Cat# TL303695, OriGene, using si‐NC as the control) or NLRP3 ORF‐clone (1:1000 dilution, Cat# RC223212, OriGene, using empty vector as the control) using Lipofectamine 3000 reagent according to the manufacturer's protocol.

Techniques: Inhibition, Transfection, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Control

Journal: Journal of Cellular and Molecular Medicine

Article Title: KIF1B Regulates NLRP3 ‐Mediated Pyroptosis in Asthma Progression

doi: 10.1111/jcmm.70975

Figure Lengend Snippet: KIF1B regulates IL‐13‐induced inflammation in BEAS‐2B cells via NLRP3. BEAS‐2B cells were transfected with si‐NC, si‐KIF1B + empty vector or si‐KIF1B + NLRP3‐ORF plasmid, with or without IL‐13 treatment. (A) NLRP3 gene expression was measured by qRT‐PCR following NLRP3‐ORF transfection to confirm successful overexpression. (B) Following transfection with si‐KIF1B with or without NLRP3‐ORF and IL‐13 treatment, intracellular ROS, MDA and GSH levels were measured. (C) Protein expression of NLRP3, cleaved caspase‐1 and cleaved GSDMD was assessed by Western blot. (D) Cytokine levels (TNF‐α, IL‐1β, IL‐18 and IL‐10) in conditioned medium were measured by ELISA. GAPDH gene was used as an internal reference for qPCR analysis, and β‐actin was used as the loading control for normalisation in Western blot. Data are presented as mean ± SD ( n = 3; *** p < 0.001 vs. control, p < 0.01, p < 0.001 vs. IL‐13 group, & p < 0.05, & p < 0.01 vs. IL‐13 + si‐KIF1B group).

Article Snippet: Upon reaching approximately 60%–70% confluency, cells were transfected with si‐KIF1B (1:1000 dilution, Cat# TL303695, OriGene, using si‐NC as the control) or NLRP3 ORF‐clone (1:1000 dilution, Cat# RC223212, OriGene, using empty vector as the control) using Lipofectamine 3000 reagent according to the manufacturer's protocol.

Techniques: Transfection, Plasmid Preparation, Gene Expression, Quantitative RT-PCR, Over Expression, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Control

Journal: Journal of Cellular and Molecular Medicine

Article Title: KIF1B Regulates NLRP3 ‐Mediated Pyroptosis in Asthma Progression

doi: 10.1111/jcmm.70975

Figure Lengend Snippet: KIF1B knockdown reduces OVA‐induced inflammation and pyroptosis in mouse pulmonary tissues. C57BL/6 mice were infected with lentiviral particles carrying sh‐KIF1B or sh‐NC, and subsequently treated with OVA for 14 days to induce asthmatic responses. Lung tissues were collected at sacrifice. (A) Wright‐Giemsa staining was used to quantify total cells, macrophages, eosinophils, lymphocytes and neutrophils in BALF. (B) IgE, TNF‐α, IL‐1β, IL‐18 and IL‐10 levels in BALF were measured by ELISA. (C) Protein levels of NLRP3, cleaved caspase‐1 and cleaved GSDMD in lung homogenates were assessed by Western blot. β‐actin was used as the loading control for normalisation in Western blot. Data are presented as mean ± SD ( n = 8; *** p < 0.001 vs. control, p < 0.01, p < 0.001 vs. OVA group).

Article Snippet: Upon reaching approximately 60%–70% confluency, cells were transfected with si‐KIF1B (1:1000 dilution, Cat# TL303695, OriGene, using si‐NC as the control) or NLRP3 ORF‐clone (1:1000 dilution, Cat# RC223212, OriGene, using empty vector as the control) using Lipofectamine 3000 reagent according to the manufacturer's protocol.

Techniques: Knockdown, Infection, Staining, Enzyme-linked Immunosorbent Assay, Western Blot, Control