Journal: Frontiers in Immunology

Article Title: Critical Role for the NLRP3 Inflammasome in Mediating IL-1β Production in Shigella sonnei -Infected Macrophages

doi: 10.3389/fimmu.2020.01115

Figure Lengend Snippet: S. sonnei induces the secretion of IL-1β, IL-18, NLRP3, ASC, and active caspase-1 in macrophages. (A) J774A.1 macrophages, THP-1 macrophages, PBMCs or BMDM were primed with 1 μg/ml LPS for 4 h and then infected with S. sonnei for an additional 20 h. The levels of IL-1β in the supernatants were measured by ELISA. (B–E) J774A.1 macrophages or THP-1 macrophages were primed with 1 μg/ml LPS for 4 h followed and then infected with S. sonnei for an additional 20 h or stimulated with 5 mM ATP for an additional 0.5 h. The levels of IL-1β (B) , IL-18 (C) , caspase-1 (D) , NLRP3, and ASC (E) in the supernatants were measured by Western blotting. The ELISA data are expressed as the mean ± SD of four separate experiments. The Western blotting results are representative of three different experiments. * and *** indicate significant differences at the levels of p < 0.05 and p < 0.001, respectively, compared to untreated control cells (one-way ANOVA with Dunnett's multiple comparisons test).

Article Snippet: Antibodies against IL-1β (AB-401-NA, polyclonal antibody) were purchased from R&D Systems (Minneapolis, MN).

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

Journal: Frontiers in Immunology

Article Title: Critical Role for the NLRP3 Inflammasome in Mediating IL-1β Production in Shigella sonnei -Infected Macrophages

doi: 10.3389/fimmu.2020.01115

Figure Lengend Snippet: S. sonnei induces IL-1β secretion through the NLRP3 inflammasome. (A,B) J774A.1 macrophages or BMDM were primed with 1 μg/ml LPS for 4 h and then treated with YVAD-CHO (A) or MCC950 (B) for 0.5 h. The cells were then infected with 50 MOI S. sonnei for an additional 20 h. The levels of IL-1β in the supernatants were measured by ELISA. (C,D) Wild-type or NLRP3 knockout J774A.1 macrophages were primed with 1 μg/ml LPS for 4 h and then infected with 50 MOI S. sonnei for an additional 20 h. The levels of IL-1β (C) and caspase-1 (D) in the supernatants were measured by ELISA and Western blotting, respectively. The ELISA data are expressed as the mean ± SD of four separate experiments. The Western blotting results are representative of three different experiments. * and *** indicate significant differences at the levels of p < 0.05 and p < 0.001, respectively, compared to S. sonnei -infected cells (A,B) or untreated control cells (C) (one-way ANOVA with Dunnett's multiple comparisons test).

Article Snippet: Antibodies against IL-1β (AB-401-NA, polyclonal antibody) were purchased from R&D Systems (Minneapolis, MN).

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Knock-Out, Western Blot, Control

Journal: Frontiers in Immunology

Article Title: Critical Role for the NLRP3 Inflammasome in Mediating IL-1β Production in Shigella sonnei -Infected Macrophages

doi: 10.3389/fimmu.2020.01115

Figure Lengend Snippet: S. sonnei activates the NLRP3 inflammasome through P 2 X 7 receptor-mediated potassium efflux. (A–C) J774A.1 macrophages were primed with 1 μg/ml LPS for 4 h and then treated with KCl (A) , glibenclamide (B) , probenecid or carbenoxolone (C) for 0.5 h. The cells were then infected with 50 MOI S. sonnei for an additional 20 h. The levels of IL-1β and caspase-1 in the supernatants were measured by ELISA and Western blotting, respectively. (D,E) Mock or P 2 X 7 knockdown J774A.1 macrophages were primed with 1 μg/ml LPS for 4 h and then infected with 50 MOI S. sonnei for an additional 20 h or stimulated with 5 mM ATP for an additional 0.5 h. The levels of IL-1β (D) and caspase-1 (E) in the supernatants were measured by ELISA and Western blotting, respectively. The levels of P 2 X 7 in mock or P 2 X 7 knockdown J774A.1 macrophages were measured by Western blotting and flow cytometry (D) . (F) Mock or P 2 X 7 knockdown J774A.1 macrophages were stimulated with 1 μg/ml LPS for 6 h. The levels of NLRP3 and proIL-1β in the cell lysates were measured by Western blotting. The ELISA data are expressed as the mean ± SD of four separate experiments. The Western blotting results are representative of three different experiments. * and *** indicate significant differences at the levels of p < 0.05 and p < 0.001, respectively, compared to S. sonnei -infected cells (A–C) or untreated control cells (D) (one-way ANOVA with Dunnett's multiple comparisons test).

Article Snippet: Antibodies against IL-1β (AB-401-NA, polyclonal antibody) were purchased from R&D Systems (Minneapolis, MN).

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

Journal: Frontiers in Immunology

Article Title: Critical Role for the NLRP3 Inflammasome in Mediating IL-1β Production in Shigella sonnei -Infected Macrophages

doi: 10.3389/fimmu.2020.01115

Figure Lengend Snippet: S. sonnei activates the NLRP3 inflammasome through H 2 O 2 production and lysosomal damage. (A) J774A.1 macrophages were primed with 1 μg/ml LPS for 4 h and then infected with 50 MOI S. sonnei for an additional 20 h. The levels of intracellular ROS were measured by CM-H 2 DCFDA staining, and the data were acquired by flow cytometry. (B) J774A.1 macrophages or BMDM were primed with 1 μg/ml LPS for 4 h and then treated with NAC for 0.5 h. The cells were then infected with 50 MOI S. sonnei for an additional 20 h. The levels of IL-1β and caspase-1 in the supernatants were measured by ELISA and Western blotting, respectively. (C) J774A.1 macrophages or BMDM were primed with 1 μg/ml LPS for 4 h and then treated with NDGA for 0.5 h. The cells were then infected with 50 MOI S. sonnei for an additional 20 h. The levels of IL-1β in the supernatants were measured by ELISA. (D,E) J774A.1 macrophages were primed with 1 μg/ml LPS for 4 h and then treated with NH 4 Cl and CQ (D) or CA-074-Me (E) for 0.5 h. The cells were then infected with 50 MOI S. sonnei for an additional 20 h. The levels of IL-1β in the supernatants were measured by ELISA. The ELISA data are expressed as the mean ± SD of four separate experiments. The flow cytometry and Western blotting results are representative of three different experiments. *, **, and *** indicate significant differences at the levels of p < 0.05, p < 0.01 and p < 0.001, respectively, compared to untreated control cells (A) or S. sonnei -infected cells (B–E) [two-tailed t -test in panel (A) ; one-way ANOVA with Dunnett's multiple comparisons test in panels (B–E) ].

Article Snippet: Antibodies against IL-1β (AB-401-NA, polyclonal antibody) were purchased from R&D Systems (Minneapolis, MN).

Techniques: Infection, Staining, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Western Blot, Control, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Critical Role for the NLRP3 Inflammasome in Mediating IL-1β Production in Shigella sonnei -Infected Macrophages

doi: 10.3389/fimmu.2020.01115

Figure Lengend Snippet: S. sonnei activates the NLRP3 inflammasome through mitochondrial damage. (A) J774A.1 macrophages were primed with 1 μg/ml LPS for 4 h and then treated with 20 μM MnTBAP for 0.5 h. The cells were then infected with 50 MOI S. sonnei for an additional 20 h. The levels of mitochondrial ROS were measured by MitoSOX staining and flow cytometry. (B) J774A.1 macrophages were primed with 1 μg/ml LPS for 4 h and then treated with MnTBAP for 0.5 h. The cells were then infected with 50 MOI S. sonnei for an additional 20 h. The levels of IL-1β in the supernatants were measured by ELISA. (C) J774A.1 macrophages were primed with 1 μg/ml LPS for 4 h and then infected with 50 MOI S. sonnei for an additional 20 h. The mitochondrial membrane potential was measured by DiOC 2 (3) staining and flow cytometry. (D) J774A.1 macrophages were primed with 1 μg/ml LPS for 4 h and then treated with cyclosporine A for 0.5 h. The cells were then infected with 50 MOI S. sonnei for an additional 20 h. The levels of IL-1β in the supernatants were measured by ELISA. The ELISA data are expressed as the mean ± SD of four separate experiments. The flow cytometry results are representative of three different experiments. ** and *** indicate significant differences at the levels of p < 0.01 and p < 0.001, respectively, compared to S. sonnei -infected cells (B,D) or as indicated (A,C) [two-tailed t -test in panels (A,C) ; one-way ANOVA with Dunnett's multiple comparisons test in panels (B,D) ].

Article Snippet: Antibodies against IL-1β (AB-401-NA, polyclonal antibody) were purchased from R&D Systems (Minneapolis, MN).

Techniques: Infection, Staining, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Membrane, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Critical Role for the NLRP3 Inflammasome in Mediating IL-1β Production in Shigella sonnei -Infected Macrophages

doi: 10.3389/fimmu.2020.01115

Figure Lengend Snippet: Phagocytosis of live S. sonnei is required for the full activation of the NLRP3 inflammasome. (A) J774A.1 macrophages were primed with 1 μg/ml LPS for 4 h and then treated with cytochalasin D for 0.5 h. The cells were then infected with 50 MOI S. sonnei for an additional 20 h. The levels of IL-1β and TNF-α in the supernatants were measured by ELISA. (B) The TLR2 mRNA expression of mock or TLR2 knockdown J774A.1 macrophages was measured by RT-qPCR. Mock or TLR2 knockdown J774A.1 macrophages were stimulated with 1 μg/ml Pam3CSK4 for 6 h. The levels of TNF-α in the supernatants were measured by ELISA. (C) Mock or TLR2 knockdown J774A.1 macrophages were primed with 1 μg/ml LPS for 4 h and then infected with 50 MOI live, heat-killed or freeze/thaw-killed S. sonnei for an additional 20 h. The levels of IL-1β and TNF-α in the supernatants were measured by ELISA. The ELISA data are expressed as the mean ± SD of four separate experiments. * and *** indicate significant differences at the levels of p < 0.05 and p < 0.001, respectively, compared to S. sonnei -infected cells (A) or live S. sonnei -infected cells (C) or as indicated (B) . ### indicates significant differences at the levels of p < 0.001 compared to sh-SC cells [one-way ANOVA with Dunnett's multiple comparisons test in panels (A,C) ; two-tailed t -test in panel (B) ].

Article Snippet: Antibodies against IL-1β (AB-401-NA, polyclonal antibody) were purchased from R&D Systems (Minneapolis, MN).

Techniques: Activation Assay, Infection, Enzyme-linked Immunosorbent Assay, Expressing, Knockdown, Quantitative RT-PCR, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Critical Role for the NLRP3 Inflammasome in Mediating IL-1β Production in Shigella sonnei -Infected Macrophages

doi: 10.3389/fimmu.2020.01115

Figure Lengend Snippet: NLRP3 knockout increases the bactericidal activity of macrophages against S. sonnei . (A,B) Wild-type or NLRP3-knockout J774A.1 macrophages were infected with 50 MOI S. sonnei for 15 min or 20 h. The cells were lysed, and the number of engulfed live S. sonnei cells was determined by the CFU assay and is indicated in the upper right corner (A) . The mean CFUs of cells infected with S. sonnei for 15 min or 20 h and the number of killed bacteria were calculated by subtracting the 20 h CFU assay result from the 15 min CFU assay result and are shown in panel (B) . The data are expressed as the mean ± SD of five separate experiments. ***indicates a significant difference at the level of p < 0.001 compared to wild-type cells (two-tailed t- test).

Article Snippet: Antibodies against IL-1β (AB-401-NA, polyclonal antibody) were purchased from R&D Systems (Minneapolis, MN).

Techniques: Knock-Out, Activity Assay, Infection, Colony-forming Unit Assay, Bacteria, Two Tailed Test

Journal: Molecules

Article Title: In Vitro Neuroprotective and Anti-Inflammatory Activities of Natural and Semi-Synthetic Spirosteroid Analogues

doi: 10.3390/molecules21080992

Figure Lengend Snippet: Anti-excitotoxic activity of compounds in glutamate-induced cell death. ( A ) PC12 cell cultures were exposed to an established excitotoxic concentration of glutamate (Glu) (20 mM), and some were also treated with each compound ( S9 and S15 semi-synthetic derivatives; E3, estriol; SL, solasodine; E1, estrone; T, testosterone; TDCA, taurodeoxycholic acid) or solvent control (DMSO). After 24 h of exposition to the damage and treatment, cellular viability was determined by the MTT assay; ( B ) Dose-response of Compound S15 in the same model of glutamate-induced excitotoxicity. Data are represented as the percent with respect to the untreated cells, representing the highest cell viability. * p < 0.05, ** p < 0.01 and *** p < 0.001, according to one-way ANOVA and Tukey’s post-hoc test.

Article Snippet: The amount of IL-1β released into the culture medium was measured using a sandwich ELISA system with antibodies specifics for rat IL-1β (Polyclonal Rat IL-1β/IL-1F2 Catalog Number AF-501-NA and Polyclonal Rat IL-1β/IL-1F2 Biotinylated Catalog Number BAF501, R&D Systems, Minneapolis, MN, USA), following the manufacturer’s instructions.

Techniques: Activity Assay, Concentration Assay, Solvent, Control, MTT Assay

Journal: Molecules

Article Title: In Vitro Neuroprotective and Anti-Inflammatory Activities of Natural and Semi-Synthetic Spirosteroid Analogues

doi: 10.3390/molecules21080992

Figure Lengend Snippet: Neuroprotective activity of compounds against cell damage induced by chemical hypoxia and glucose deprivation. PC12 cells were cultured in oxygen and glucose deprivation conditions (OGD), specifically in the absence of glucose and in the presence of 600 µM of potassium cyanide (KCN) for 1 h, and then, normal culture medium was reperfused to the cells. After 4 h of the reperfusion start, some PC12 cells were treated with solvent control (DMSO), solasodine (SL) or different concentrations of the S15 derivative. After 20 h of exposition to the reperfusion damage and treatment, cellular viability was determined by the MTT assay. Data are represented as the percent with respect to the untreated cells, representing the highest cell viability. Asterisks indicate statistical differences between the treatment and respective control condition (*** p < 0.001) according to one-way ANOVA and Tukey’s post-hoc test.

Article Snippet: The amount of IL-1β released into the culture medium was measured using a sandwich ELISA system with antibodies specifics for rat IL-1β (Polyclonal Rat IL-1β/IL-1F2 Catalog Number AF-501-NA and Polyclonal Rat IL-1β/IL-1F2 Biotinylated Catalog Number BAF501, R&D Systems, Minneapolis, MN, USA), following the manufacturer’s instructions.

Techniques: Activity Assay, Cell Culture, Solvent, Control, MTT Assay

Journal: Molecules

Article Title: In Vitro Neuroprotective and Anti-Inflammatory Activities of Natural and Semi-Synthetic Spirosteroid Analogues

doi: 10.3390/molecules21080992

Figure Lengend Snippet: In vitro anti-inflammatory activity of the S15 derivative in activated primary microglia. Microglia-enriched primary cultures were obtained from brains of adult male rats. Different concentrations of the S15 derivative, solvent control (DMSO) or 10 µM of dexamethasone (DX) as the positive control of inhibition were applied to some microglia cultures, 30 min before the activation of microglia cells with lipopolysaccharide (LPS, 1 µg/mL) and murine recombinant interferon gamma (IFNγ, 20 U/mL). Untreated (UT) microglia cells were included as the negative control of inhibition. After 24 h of exposition or not to the stimulus and treatment, the IL-1β level was quantified by ELISA ( A ); and the content of nitrite was determined by the Griess assay ( B ). Asterisks indicate statistical differences between the treatment and respective control condition (* p < 0.05, ** p < 0.01 and *** p < 0.001) according to one-way ANOVA and Tukey’s post-hoc test.

Article Snippet: The amount of IL-1β released into the culture medium was measured using a sandwich ELISA system with antibodies specifics for rat IL-1β (Polyclonal Rat IL-1β/IL-1F2 Catalog Number AF-501-NA and Polyclonal Rat IL-1β/IL-1F2 Biotinylated Catalog Number BAF501, R&D Systems, Minneapolis, MN, USA), following the manufacturer’s instructions.

Techniques: In Vitro, Activity Assay, Solvent, Control, Positive Control, Inhibition, Activation Assay, Recombinant, Negative Control, Enzyme-linked Immunosorbent Assay, Griess Assay

Journal: PLoS Pathogens

Article Title: Functional and Evolutionary Analyses Identify Proteolysis as a General Mechanism for NLRP1 Inflammasome Activation

doi: 10.1371/journal.ppat.1006052

Figure Lengend Snippet: ( A ) The amino acid sequence of the first 244 residues of NLRP1B B6 was aligned to the homologous sequence of NLRP1B 129 . The arrow above the alignment indicates the LF-cleavage site in NLRP1B 129 . Asterisks indicate sites of amino acid identity. ( B ) For detection of NLRP1B expression, 293T cells were transfected with the indicated amounts of and empty vector (V) or plasmids encoding GFP-HA-NLRP1B 129 or GFP-HA-NLRP1B B6 (construct schematics and the predicted molecular weight of each protein is depicted in the upper panel). Cells were treated overnight with anthrax lethal toxin protein (LeTx, 1μg/ml) 24h post-transfection, and then lysates were analyzed by immunoblotting (IB) with the indicated antibodies. For NLRP1 expression and cleavage, CASP1 and IL1B were omitted to prevent cell death and resulting apparent differences of expression. For blots probed with anti-HA (NLRP1B), the lysates were not boiled prior to loading to prevent aggregation and smearing of full-length and FIIND-processed NLRP1B on the immunoblot. To visualize the N-terminal processed form of NLRP1B, the lysates were boiled and resolved on a separate gel. ( C ) 293T Cells were transfected with the same amount of titrated NLRP1B encoding plasmid and treated as in B, but transfections also included plasmids encoding mouse CASP1 (200ng), IL-1β (200ng) and 200ng of empty vector. ( D ) For detection of NLRP1B expression, 293T cells were transfected for 36h with 250ng of plasmids encoding WT 129 or B6 NLRP1B or mutants engineered to express the TEV-protease site. Each plasmid was co-transfected with either 100ng of empty vector (V) or plasmids encoding TEV-protease (pTEV) or lethal factor protease (pLF), supplemented with 300ng empty vector (pMSCV). Cells were not treated with LeTx and lysates were analyzed by immunoblotting as in (B). ( E ) Cells were treated as in C, but with 8ng of plasmids encoding NLRP1B, along with 200ng of plasmids encoding mCASP-1 and mIL-1β, and 200ng empty vector to normalize plasmid quantities. Lower quantities of NLRP1B were transfected as compared to panel D so to avoid spontaneous NLPR1B activation. For panels B-E, data shown are representative of at least three similar experiments.

Article Snippet: The following antibodies were used for the following antigens: HA mAB 3F10 (Roche), MEK-2 SC-13115 (Santa Cruz), MYC mAb 9E10 (Clonetech), IL-1β AF-401-NA (R&D systems).

Techniques: Sequencing, Expressing, Transfection, Plasmid Preparation, Construct, Molecular Weight, Western Blot, Activation Assay

Journal: PLoS Pathogens

Article Title: Functional and Evolutionary Analyses Identify Proteolysis as a General Mechanism for NLRP1 Inflammasome Activation

doi: 10.1371/journal.ppat.1006052

Figure Lengend Snippet: ( A ) The amino acid sequence of the first 244 residues of NLRP1A B6 was aligned to NLRP1B 129 and NLRP1B B6 . The arrow above the alignment indicates the LF-cleavage site in NLRP1B 129 . ( B ) 293T cells were transfected for 36h with 200ng empty vector (V) or plasmids pcDNA3.1-HA-NLRP1B 129 or -NLRP1A B6 -MYC (CMV promoter) along with 200ng mCASP1 and 200ng mIL-1β expression vectors. Cells were treated overnight with anthrax lethal toxin (LeTx, 1μg/ml) 24h post-transfection, and then lysates were analyzed by immunoblotting (IB) with the indicated antibodies. ( C ) 293T cells were transfected for 36h with plasmids encoding 400ng GFP-HA-NLRP1B 129 or GFP-HA-NLRP1A B6 (under the control of the LTR promoter in pMSCV) or with mutants engineered to contain an N-terminal TEV protease site. Cells were also co-transfected with 100ng empty vector (V) or plasmids encoding TEV-protease (pTEV) or lethal factor protease (pLF), plus 300ng empty pMSCV. ( D ) To assess IL-1β cleavage, cells were transfected as in C, but with 8ng of GFP-HA-NLRP1B and co-transfected with vectors encoding 200ng mCASP1 and 200ng mIL-1β. For panels B-D, data shown are representative of at least three similar experiments.

Article Snippet: The following antibodies were used for the following antigens: HA mAB 3F10 (Roche), MEK-2 SC-13115 (Santa Cruz), MYC mAb 9E10 (Clonetech), IL-1β AF-401-NA (R&D systems).

Techniques: Sequencing, Transfection, Plasmid Preparation, Expressing, Western Blot, Control

Journal: PLoS Pathogens

Article Title: Functional and Evolutionary Analyses Identify Proteolysis as a General Mechanism for NLRP1 Inflammasome Activation

doi: 10.1371/journal.ppat.1006052

Figure Lengend Snippet: ( A ) For detection of NLRP1 expression, 293T cells were co-transfected for 24h with 100ng human full-length pcDNA3.1-NLRP1-MYC (CMV promoter) (FL), or with constructs in which the leucine rich repeat or pyrin domains had been deleted (ΔLRR, ΔPYD). Cells were also co-transfected with 500ng empty pcDNA3.1 vectors and with plasmids encoding human ASC (1ng), hCASP1 (1ng), and IL-1β–V5 (200ng). After 36h, cells were analyzed by immunoblot (IB) using an antibody against MYC. ( B ) Cells were transfected as in A, but to avoid spontaneous NLRP1 activation, the amount of hNLRP1 plasmid was reduced to 10ng and adjusted with 90ng of empty vector. Lysates were blotted with an anti-V5 antibody. ( C ) Cleavage sites for TEV protease were introduced into pcDNA3.1-hNLRP1-MYC at the indicated positions (see for details) to generate TEV T1, TEV T2, and TEV T3 expression constructs. 293T cells were co-transfected with 10g of these NLRP1 constructs along with 50ng of a vector encoding TEV-protease (pTEV) or a control empty vector (V), and all supplemented with 200ng empty pcDNA3.1. After 24h, lysates were analyzed by immunoblot using anti-Myc antibody to detect full-length NLRP1, the FIIND-processed (30kDa) C-terminal product, or the products that result from TEV cleavage (144kDa for T1, 155kDa for T2, and 133kDa for T3). ( D ) 293T cells were co-transfected with 5ng of NLRP1 constructs along with 50ng of vector encoding TEV-protease (pTEV) or a control empty vector (V), and plasmids encoding human ASC (1ng), CASP1 (1ng), and IL-1β-V5 (200ng), supplemented with 500ng empty pcDNA3.1. After 24h, lysates were analyzed by immunoblot using anti-V5 antibody to detect CASP1-dependent processing of IL-1β into the mature p17 fragment. ( E ) A panel of constructs for expressing full-length or truncated GFP-fused human NLRP1 in pMSCV (LTR promoter) was generated as depicted. The TEV T1 and T2 sites were introduced into two of the constructs as indicated (also see ). 293T cells were transfected with 100ng these constructs along with 50ng empty vector or a plasmid encoding the TEV protease (pTEV). Cell lysates were analyzed by immunoblot (IB) with anti-GFP. ( F ) 293T cells were transfected for 24h with 12.5ng plasmids encoding full-length or truncated GFP-fused human NLRP1 in pMSCV (LTR promoter), along with plasmids encoding human ASC (1ng), CASP1 (1ng), V5-tagged IL-1β (200ng) and TEV protease (50ng, pTEV). After 24h, lysates were analyzed by immunoblot using anti-V5 antibody to detect CASP1-dependent processing of IL-1β into the mature p17 fragment. For all panels, data shown are representative of at least three similar experiments.

Article Snippet: The following antibodies were used for the following antigens: HA mAB 3F10 (Roche), MEK-2 SC-13115 (Santa Cruz), MYC mAb 9E10 (Clonetech), IL-1β AF-401-NA (R&D systems).

Techniques: Expressing, Transfection, Construct, Western Blot, Activation Assay, Plasmid Preparation, Control, Generated

Journal: PLoS Pathogens

Article Title: Functional and Evolutionary Analyses Identify Proteolysis as a General Mechanism for NLRP1 Inflammasome Activation

doi: 10.1371/journal.ppat.1006052

Figure Lengend Snippet: ( A ) 293T cells were transfected with 100ng of CMSCV empty vector (V) or plasmids encoding mouse wild type HA-NLRP1B 129 (WT) or mutant HA-NLRP1B 129 either containing the K137R mutation in the Walker A (WA) ATP-binding site or the constitutively active ΔLRR mutant. The cells were co-transfected with vectors encoding mouse CASP1 and IL-1β. Cells expressing mouse NLRP1B were treated with LeTx for 16h where indicated. Non-boiled lysates were subject to immunoblot (IB) with anti-HA antibody, whereas boiled lysates were subject to immunoblot with anti-IL-1β to detect CASP1-dependent processing of IL-1β into the mature p17 fragment. ( B ) 239T cells were transfected with 100ng empty vector (V) or plasmids encoding hNLRP1-MYC wild-type (WT), Walker A mutant 1 K340A (WA1), Walker A mutant 2 K340R (WA2) or ΔLRR expression plasmids. The transfection complexes also contained 200ng mCASP, 200ng mIL-1β and 1ng hASC expression vectors. For both A and B, data shown are representative of at least three similar experiments.

Article Snippet: The following antibodies were used for the following antigens: HA mAB 3F10 (Roche), MEK-2 SC-13115 (Santa Cruz), MYC mAb 9E10 (Clonetech), IL-1β AF-401-NA (R&D systems).

Techniques: Transfection, Plasmid Preparation, Mutagenesis, Binding Assay, Expressing, Western Blot

Journal: Scientific Reports

Article Title: Pioglitazone Modulates the Vascular Contractility in Hypertension by Interference with ET-1 Pathway

doi: 10.1038/s41598-019-52839-6

Figure Lengend Snippet: Contribution of NFκB to the ET-1-induced oxidative stress and COX-2 expression in VSMC from hypertensive rats. Effect of lactacystin (Lac, 10 µM) on NOX-1 mRNA levels ( a ), NADPH oxidase activity ( b ), COX-2 protein expression ( c ) and mRNA levels ( d ) induced by ET-1 (0.1 µM, 1 h). ( e ) Effect of ET-1 (0.1 µM, 45 min) on nuclear p65 NFκB protein expression in VSMC from SHR; a representative blot of the cytosolic (Cy) and nuclear (Nu) expression is shown below; TATA-binding protein (TBP) and GAPDH (after reblotting) cytosolic and nuclear expressions are also shown to guarantee the successful cellular fractioning. ( f ) Representative photomicrographs of p65 NFκB immunofluorescence (red) in VSMC from SHR in control and after incubation with ET-1 (0.1 µM, 45 min, n = 7). Negative controls without primary or secondary antibody are also shown. Bar scale represents 50 µm. Statistical analysis by Student’s t-test; * P < 0.05 vs control and # P < 0.05 vs ET-1. n denotes number of experiments. Full-length blots are presented in Supplementary Fig. .

Article Snippet: Proteins were separated by 10% SDS-PAGE and transferred to polyvinyl difluoride membranes that were incubated with rabbit polyclonal antibodies for COX-2 (1:250; Cayman Chemical, Ann Arbor, MI, USA, catalogue number 160106, lot 0439169–1), NOX-1 (1:1,000; Abcam, Cambridge, UK, catalogue number ab131088, lot GR3244226-4), p65 (1:1,000, Santa Cruz Biotechnology, Santa Cruz, CA, USA, catalogue number sc-372, lot BO513) or p-JNK1/2 (1:1,000, Cell Signaling Technology, Danvers, MA, USA, catalogue number #9251, lot 24).

Techniques: Expressing, Activity Assay, Binding Assay, Immunofluorescence, Control, Incubation

Journal: Scientific Reports

Article Title: Pioglitazone Modulates the Vascular Contractility in Hypertension by Interference with ET-1 Pathway

doi: 10.1038/s41598-019-52839-6

Figure Lengend Snippet: Effect of pioglitazone on the role of NFκB and AP-1 to the ET-1-induced COX-2 expression in VSMC from hypertensive rats. ( a ) Effect of pioglitazone (Pio, 10 µM, 18 h) on ET-1 (0.1 µM, 45 min)-induced nuclear p65 NFκB protein expression in vascular smooth muscle cells (VSMC) from SHR; the representative blot of the cytosolic (Cy) and nuclear (Nu) expression is shown in Fig. . ( b ) Representative photomicrographs of p65 NFκB immunofluorescence (red) in VSMC from SHR after incubation with ET-1 (0.1 µM, 45 min) in the absence and in the presence of pioglitazone. Bar scale represents 50 µm. Effect of pioglitazone on ( c ) p-JNK1/2 protein expression (a representative blot is shown in the upper pane; lower panel shows reblotting of upper panel with JNK2 antibody) and ( d ) c-jun mRNA levels induced by ET-1 (0.1 µM, 15 min for p-JNK2 and 1 h for c-jun). Statistical analysis by Student's t-test. n denotes number of experiments. Full-length blots are presented in Supplementary Fig. .

Article Snippet: Proteins were separated by 10% SDS-PAGE and transferred to polyvinyl difluoride membranes that were incubated with rabbit polyclonal antibodies for COX-2 (1:250; Cayman Chemical, Ann Arbor, MI, USA, catalogue number 160106, lot 0439169–1), NOX-1 (1:1,000; Abcam, Cambridge, UK, catalogue number ab131088, lot GR3244226-4), p65 (1:1,000, Santa Cruz Biotechnology, Santa Cruz, CA, USA, catalogue number sc-372, lot BO513) or p-JNK1/2 (1:1,000, Cell Signaling Technology, Danvers, MA, USA, catalogue number #9251, lot 24).

Techniques: Expressing, Immunofluorescence, Incubation

Journal: International journal of molecular sciences

Article Title: Interleukin-6 Modulates the Expression and Function of HCN Channels: A Link Between Inflammation and Atrial Electrogenesis.

doi: 10.3390/ijms252212212

Figure Lengend Snippet: Figure 3. IL6 downregulates HCN channel proteins in HL1 cells. HL1 cells were grown in normal medium with (mIL6) or without (CTR) mIL6 (50 ng/mL) for 24 h. Proteins were analyzed by Western blot analysis. Panels show the representative immunoblots and the densitometric analysis of HCN1 (A), HCN2 (B), HCN4 (C), and MiRP1 (D) proteins. Data are expressed as mean ± standard error of 6 independent experiments. * p < 0.05, *** p < 0.001, p > 0.05 for HCN2/Mirp-1 CTR vs. mIL6. Unpaired t test.

Article Snippet: Blots were incubated overnight at 4 ◦C with a specific antibody against HCN1 (rabbit, #APC-056, Lot APC056AG0740, Alomone Labs, Jerusalem, Israel), HCN2 (rabbit, #APC-030, Lot APC030AG0940, Alomone Labs, Jerusalem, Israel), HCN4 (rabbit, #APC052, Lot APC052AG11120, Alomone Labs, Jerusalem, Israel), MiRP1 (rabbit, #APC-054, Lot AN-03, Alomone Labs, Jerusalem, Israel), p-STAT3Tyr705 (mouse, #4113S, Lot 6, Cell Signaling Technology, Leiden, The Netherland), STAT3 (rabbit, #4904S, Lot 7, Cell Signaling Technology, Leiden, The Netherland), and GAPDH (rabbit, #5174S, Lot 8, Cell Signaling Technology, Leiden, The Netherland).

Techniques: Western Blot

Journal: International journal of molecular sciences

Article Title: Interleukin-6 Modulates the Expression and Function of HCN Channels: A Link Between Inflammation and Atrial Electrogenesis.

doi: 10.3390/ijms252212212

Figure Lengend Snippet: Figure 6. Acute hIL6 decreases HCN4-mediated current. Current was recorded in HEK cells stably expressing human HCN4 channels. (A) Voltage clamp protocol (top panel) and representative recordings (bottom panels) in control conditions (CTR) and after exposure to 0.05 and 50 ng/mL hIL6. (B) HCN4-current activation curves in control conditions (CTR) and after exposure to hIL6 (from 0.05 to 50 ng/mL). Values are expressed as mean of 4–7 independent experiment ± standard error of the mean. $ p < 0.05 CTR vs. hIL6 0.05 to 50 ng/mL. Two-way-ANOVA followed by Sidak’s multiple comparison test. A detailed statistical analysis of data in the graph is reported in Supplementary Table S2. (C) Concentration-current curve of normalized HCN4 current amplitude measured at −80 and −120 mV plotted versus log hIL6 concentration and fitted by Hill equation.

Article Snippet: Blots were incubated overnight at 4 ◦C with a specific antibody against HCN1 (rabbit, #APC-056, Lot APC056AG0740, Alomone Labs, Jerusalem, Israel), HCN2 (rabbit, #APC-030, Lot APC030AG0940, Alomone Labs, Jerusalem, Israel), HCN4 (rabbit, #APC052, Lot APC052AG11120, Alomone Labs, Jerusalem, Israel), MiRP1 (rabbit, #APC-054, Lot AN-03, Alomone Labs, Jerusalem, Israel), p-STAT3Tyr705 (mouse, #4113S, Lot 6, Cell Signaling Technology, Leiden, The Netherland), STAT3 (rabbit, #4904S, Lot 7, Cell Signaling Technology, Leiden, The Netherland), and GAPDH (rabbit, #5174S, Lot 8, Cell Signaling Technology, Leiden, The Netherland).

Techniques: Stable Transfection, Expressing, Control, Activation Assay, Comparison, Concentration Assay

Journal: International Journal of Molecular Sciences

Article Title: Functional siRNA Delivery via Jet Nebulization: Proof-of-Concept IL-1ß Silencing in Macrophage-like THP-1 Cells

doi: 10.3390/ijms27062915

Figure Lengend Snippet: Evaluation of siRNA cytotoxicity and silencing efficiency. ( A ) XTT assay results indicating cell viability ( left ) and LDH assay results measuring cytotoxicity ( right ) in THP-1 Mφ treated with 40 nM of siRNA candidates (siIL-1β.1, siIL-1β.2, siIL-1β.3) or controls (transfection medium Opti-MEM, transfection reagent RNAiMAX, and siCon). ( B ) Schematic of the psiCHECK™-2 dual-luciferase reporter plasmid. Renilla luciferase (Rluc) serves as the primary reporter, with siRNA target sequences (TS) cloned downstream of the reporter gene. Firefly luciferase (Fluc) is co-expressed for normalization. ( C ) Relative Ren/Luc activity in HeLa cells co-transfected with siRNAs at 20 nM ( left ) or 40 nM ( right ). Relative luciferase activity was normalized to siCon set as 100%. Data are represented as mean ± SD from three independent experiments (n = 3), with statistical significance denoted as *** p ≤ 0.001, **** p ≤ 0.0001, and ns (non-significant, p > 0.05). Statistical analysis was performed using one-way ANOVA followed by Dunnett’s multiple comparison test.

Article Snippet: The membrane was incubated overnight at 4 °C with a primary antibody against IL-1β (12242, 1:1000, Cell Signaling Technologies, Danvers, MA, USA) diluted in blocking buffer.

Techniques: XTT Assay, Lactate Dehydrogenase Assay, Transfection, Luciferase, Plasmid Preparation, Clone Assay, Activity Assay, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Functional siRNA Delivery via Jet Nebulization: Proof-of-Concept IL-1ß Silencing in Macrophage-like THP-1 Cells

doi: 10.3390/ijms27062915

Figure Lengend Snippet: IL-1β protein knockdown in differentiated THP-1 cells post-siRNA transfection. Cells were treated with 100 ng/mL PMA for 72 h to induce differentiation into THP-1 Mφ. Following differentiation, cells were stabilized in PMA-free medium for 24 h before transfection with 20 nM siRNA. At 43 h post-transfection, cells were stimulated with 100 ng/mL LPS for 5 h to induce an immune response. Non-stimulated, non-transfected THP-1 Mφ served as the control condition. ( A ) Representative Western blot showing pro-IL-1β protein expression under various treatment conditions, including the addition (+) or absence (−) of PMA, LPS, and siRNA transfection. β-actin served as a loading control to confirm equal protein loading (20 μg per lane). ( B ) Densitometric quantification of pro-IL-1β protein levels from Western blots, normalized to β-actin and expressed relative to siCon set as 100%. ( C ) Relative IL-1β protein levels quantified by ELISA, normalized to siCon set as 100%. Data are presented as mean ± SD from three independent experiments (n = 3), with statistical significance denoted as **** p ≤ 0.0001. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s multiple comparison test.

Article Snippet: The membrane was incubated overnight at 4 °C with a primary antibody against IL-1β (12242, 1:1000, Cell Signaling Technologies, Danvers, MA, USA) diluted in blocking buffer.

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

Journal: International Journal of Molecular Sciences

Article Title: Functional siRNA Delivery via Jet Nebulization: Proof-of-Concept IL-1ß Silencing in Macrophage-like THP-1 Cells

doi: 10.3390/ijms27062915

Figure Lengend Snippet: Evaluation of cell viability and nebulization setup for siRNA delivery. ( A ) Custom nebulizer chamber for siRNA delivery, incorporating the PARI SINUS2 nebulizer with 3D-printed adapters for a 6-well plate. The setup includes an aspirator outlet to prevent mist leakage. ( B ) XTT assay results for THP-1 Mφ: ( Top ) Viability after removal of medium for varying time points up to 30 min. ( Bottom ) Viability after nebulization with Opti-MEM for up to 15 min. ( C ) Live-dead staining of THP-1 Mφ: ( Top ) Control cells cultured without nebulization. ( Bottom ) Cells nebulized with 20 nM siRNA (siIL-1β.2) for 5 min. Live cells are indicated in green and dead cells in red. Data are represented as mean ± SD from three independent experiments (n = 3), ns indicates a non-significant difference ( p > 0.05). Statistical analysis was performed using one-way ANOVA followed by Dunnett’s multiple comparison test. Scale bars = 200 μm.

Article Snippet: The membrane was incubated overnight at 4 °C with a primary antibody against IL-1β (12242, 1:1000, Cell Signaling Technologies, Danvers, MA, USA) diluted in blocking buffer.

Techniques: XTT Assay, Staining, Control, Cell Culture, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Functional siRNA Delivery via Jet Nebulization: Proof-of-Concept IL-1ß Silencing in Macrophage-like THP-1 Cells

doi: 10.3390/ijms27062915

Figure Lengend Snippet: Evaluation of siRNA delivery and functionality post-nebulization. ( A ) Fluorescence microscopy of THP-1 Mφ transfected with fluorescent-labeled siCon.Cy3 via nebulization. Shown are DAPI nuclear staining (blue), phase contrast, siCon.Cy3 signal (red), and the merged overlay. ( B ) Dual-luciferase reporter assay after sequential transfection, in which THP-1 Mφ were first transfected with the reporter plasmid and subsequently with siRNA 6 h later. Relative luciferase activity was normalized to siCon set as 100%. ( C ) Cell viability assessment by XTT assay in HeLa cells following temporary medium removal for up to 30 min. ( D ) Dual-luciferase reporter assay following siRNA delivery via nebulization, normalized to siCon set as 100%. Data are presented as mean ± SD from three independent experiments (n = 3), with statistical significance denoted as **** p ≤ 0.0001 and ns, non-significant ( p > 0.05). Statistical analysis was performed using one-way ANOVA with Dunnett’s multiple comparisons or unpaired Student’s t -test. Scale bars = 20 μm.

Article Snippet: The membrane was incubated overnight at 4 °C with a primary antibody against IL-1β (12242, 1:1000, Cell Signaling Technologies, Danvers, MA, USA) diluted in blocking buffer.

Techniques: Fluorescence, Microscopy, Transfection, Labeling, Staining, Luciferase, Reporter Assay, Plasmid Preparation, Activity Assay, XTT Assay

Journal: International Journal of Molecular Sciences

Article Title: Functional siRNA Delivery via Jet Nebulization: Proof-of-Concept IL-1ß Silencing in Macrophage-like THP-1 Cells

doi: 10.3390/ijms27062915

Figure Lengend Snippet: Validation of nebulized siRNA for endogenous IL-1β knockdown in differentiated THP-1 cells. Cells were treated with 100 ng/mL PMA for 72 h to induce differentiation into THP-1 Mφ. Following differentiation, cells were cultured in PMA-free medium for 24 h before transfection with 20 nM siRNA using the PARI SINUS2 Nebulizer System. At 43 h post-transfection, cells were stimulated with 100 ng/mL LPS for 5 h to induce an immune response. Non-stimulated, non-transfected THP-1 Mφ served as the control condition. ( A ) Representative Western blot showing pro-IL-1β protein expression under various treatment conditions, including PMA-induced differentiation, LPS stimulation, and siRNA nebulization. The presence or absence of the respective treatment is indicated as + and −. β-actin served as a loading control to confirm equal protein loading (20 μg per lane). ( B ) Densitometric quantification of pro-IL-1β protein levels from Western blots, normalized to β-actin and expressed relative to siCon (set to 100%). ( C ) Relative IL-1β protein levels quantified by ELISA, normalized to siCon set as 100%. Data are presented as mean ± SD from three independent experiments (n = 3), with statistical significance denoted as **** p ≤ 0.0001. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s multiple comparison test.

Article Snippet: The membrane was incubated overnight at 4 °C with a primary antibody against IL-1β (12242, 1:1000, Cell Signaling Technologies, Danvers, MA, USA) diluted in blocking buffer.

Techniques: Biomarker Discovery, Knockdown, Cell Culture, Transfection, Control, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Experimental & Molecular Medicine

Article Title: Discovery of ETI41 and ETI60: novel selective endosomal Toll-like receptor inhibitors for the treatment of autoimmune diseases

doi: 10.1038/s12276-025-01526-w

Figure Lengend Snippet: a , b Western blot analysis was used to quantify the protein levels of phosphorylated p-ERK, p-JNK, p38, Iκ-Bα and p65. RAW 264.7 cells were treated with 5 μM or 10 μM of ETI41 or ETI60, followed by exposure to IMQ (2 μg/ml) for 20 or 30 min ( a ) and ODN2395 (1 μM) for 40 or 50 min ( b ) respectively. β-Actin served as a loading control for the cytosol, while lamin A/C was utilized as a loading control for the nucleus. c Protein levels of IRF7 were measured after treatment with 5 μM of ETI41 or ETI60, followed by treatment with ODN2395 (1 μM) for 6 or 8 h. d BMDC cells from ex vivo were used to analyze mRNA expression levels of IL-12p40 , IFN-β and CD40 . Cells were pretreated with ETI41 (0.2 and 1 μM), followed by treatment with ODN2395 (1 μM) for 3 h. All experiments were independently conducted three times. Statistical differences between the induced and other cases were analyzed and verified using a one-tailed Student’s t -test (* P < 0.05, ** P < 0.01, *** P < 0.001). e – g The TLR7 agonist IMQ (1 μM) was used in 3’ RNA-seq analysis to induce signaling associated with immune responses in RAW 264.7 cells. RNA-seq analysis showed that ETI41 and ETI60 at a concentration of 10 μM significantly changed gene expression patterns (fold change >2, P < 0.05) in various categories after 2 or 4 h of IMQ treatment. ( e – g ) depict changes in gene expression categories without pretreatment ( e ) after pretreatment with ETI41 ( f ) or after pretreatment with ETI60 ( g ) followed by IMQ stimulation. h Heatmaps depicting gene expression patterns within the inflammation and immune response categories (fold change >2.5, P < 0.05) of ETI41 or ETI60, dividing the time after IMQ treatment into 0, 2 and 4 h, respectively. Values of each fold change compared with the noninduced case are described, with upregulated genes shown in red and downregulated genes shown in blue.

Article Snippet: Phospho-SAPK/JNK (9251S, lot no. 27), phospho-p38 MAPK (9211S, lot no. 26), IκB-α (9242S, lot no. 11), ERK 1/2 (9102S, lot no. 27), IRF-7 (72073S, lot no. 1), NF-κB p65 (6956S, lot no. 10), SAPK/JNK (9252S, lot no. 18), p38 MAPK (9212S, lot no. 27) are purchased from Cell Signaling Technology Inc. Phospho-ERK 1/2 (sc-81492, lot no. 1123), β-actin (sc47778, lot no. H1924) and Lamin A/C (sc-20681, lot no. 131913) are purchased from Santa Cruz Biotechnology.

Techniques: Western Blot, Control, Ex Vivo, Expressing, One-tailed Test, RNA Sequencing, Concentration Assay, Gene Expression