Journal: Innate immunity

Article Title: Myeloid-derived NF-κB negative regulation of PU.1 and cEBPβ-driven pro-inflammatory cytokine production restrains LPS-induced Shock

doi: 10.1177/1753425916681444

Figure Lengend Snippet: (A). U-LPS (E. coli K12)–challenged survival of p65Δmye and littermate WT mice. Serum cytokine levels of B) IL-1β, C) IL-6, and D) TNF-α at indicated time points (hours) in WT and p65Δmye mice following one i.p. injection with U-LPS (E. coli K12) (40 mg/kg). Data represent the mean ± SEM of 2 independent experiments with (A). n = 7–11 mice per group and (B – D). n = 3–11 mice per group. (*p < 0.05) between groups.

Article Snippet: The following antibodies were used: rabbit anti-mouse caspase-1 p10 (M20; Santa Cruz; Santa Cruz, CA) and goat anti-mouse IL-1β/IL-1F2 (R&D Systems; Minneapolis, Minn) followed by goat anti-rabbit (Calbiochem; Darmstadt, Germany) or sheep anti-goat (Jackson ImmunoResearch Laboratories; West Grove, PA) peroxidase-conjugated antibodies.

Techniques: Injection

Journal: Innate immunity

Article Title: Myeloid-derived NF-κB negative regulation of PU.1 and cEBPβ-driven pro-inflammatory cytokine production restrains LPS-induced Shock

doi: 10.1177/1753425916681444

Figure Lengend Snippet: A and B, Flow cytometry analyses of F4/80, CD11b, and Ly6C expression in WT and p65Δmye BMDMs, respectively. Panel labels of a-d represent indicated gatings. C, Pro-inflammatory cytokine IL-1β, IL-6, and TNF-α secretion from BMDMs pretreated for 0, 3, or 6 h with 100 ng/mL U-LPS (E. coli K12) followed by 1 h of 2 mM ATP stimulation. D, Viability of BMDMs determined by MTT assay. Cells were treated with 0, 1, 10 100, or 1000 ng/mL U-LPS (E. coli K12) for 24 h prior to MTT assay. Data represent the mean ± SEM from n = 2–3 separate experiments. Significant differences indicated (*p < 0.05; **p < 0.01; ***p < 0.005) between groups. N.D., not detectable.

Article Snippet: The following antibodies were used: rabbit anti-mouse caspase-1 p10 (M20; Santa Cruz; Santa Cruz, CA) and goat anti-mouse IL-1β/IL-1F2 (R&D Systems; Minneapolis, Minn) followed by goat anti-rabbit (Calbiochem; Darmstadt, Germany) or sheep anti-goat (Jackson ImmunoResearch Laboratories; West Grove, PA) peroxidase-conjugated antibodies.

Techniques: Flow Cytometry, Expressing, MTT Assay

Journal: Innate immunity

Article Title: Myeloid-derived NF-κB negative regulation of PU.1 and cEBPβ-driven pro-inflammatory cytokine production restrains LPS-induced Shock

doi: 10.1177/1753425916681444

Figure Lengend Snippet: A, Pro-inflammatory cytokine IL-1β, IL-6, and TNF-α secretion; B, Il1b and caspase-1 gene expression analyzed by quantitative real-time PCR; C, Western blot of pro-IL-1β expression in unstimulated peritoneal inflammatory MΦs. D, Western blot of pro-IL-1β, IL-1β, pro-caspase-1, and caspase-1 expression of peritoneal MΦs pretreated for 6 h with 0, 10, or 100 ng/mL U-LPS (E. coli K12) followed by a 1-h stimulation with 2 mM ATP. Data represent the mean ± SEM of 2 independent experiments with n = 2–3 mice per group. Cytokine expression is normalized to the expression of the reference gene (hprt). Significant differences (*p < 0.05; **p < 0.01; ***p < 0.005) between groups. N.D., not detectable.

Article Snippet: The following antibodies were used: rabbit anti-mouse caspase-1 p10 (M20; Santa Cruz; Santa Cruz, CA) and goat anti-mouse IL-1β/IL-1F2 (R&D Systems; Minneapolis, Minn) followed by goat anti-rabbit (Calbiochem; Darmstadt, Germany) or sheep anti-goat (Jackson ImmunoResearch Laboratories; West Grove, PA) peroxidase-conjugated antibodies.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot

Journal: Frontiers in endocrinology

Article Title: Excessive Iodine Promotes Pyroptosis of Thyroid Follicular Epithelial Cells in Hashimoto's Thyroiditis Through the ROS-NF-κB-NLRP3 Pathway.

doi: 10.3389/fendo.2019.00778

Figure Lengend Snippet: FIGURE 1 | Aberrant pyroptosis occurs in the thyroid tissues of HT patients and is induced by excessive iodine in vitro. (A,B) Gasdermin D (GSDMD) protein levels in the thyroid tissue of HT patients (n = 20) and controls (n = 10) were analyzed by immunoblots. “Control” indicates tissues from patients with nodular goiter of the thyroid. Representative immunoblotting results and quantification of GSDMD are shown. (C,D) Representative results of GSDMD immunohistochemical staining in HT tissues (n = 20) and control tissues (n = 10) are shown. Brown regions represent positive expression (original magnification, ×200; scale bars, 100 µm). (E–G) Nthy-ori3-1 cells were harvested after treatment with a gradient of concentrations of sodium iodide (NaI) for 24 h. The images presented are immunoblots probed for GSDMD (Hsp60 served as the loading control). All statistical results shown are representative of three replicates. (H) The cell viability of Nthy-ori 3-1 cells was assessed by CCK-8 assays after NaI treatment for 24 h. All statistical results shown are representative of three replicates. Significant differences and P-values were calculated by unpaired t-tests or one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Thyroid sections were blocked with 2% bovine serum albumin in PBS for 30min and then incubated with mouse anti-human GSDMD or mouse antihuman IL-1β antibodies (Santa Cruz, NJ, USA) at 4◦C overnight.

Techniques: In Vitro, Western Blot, Control, Immunohistochemical staining, Staining, Expressing, CCK-8 Assay

Journal: Frontiers in endocrinology

Article Title: Excessive Iodine Promotes Pyroptosis of Thyroid Follicular Epithelial Cells in Hashimoto's Thyroiditis Through the ROS-NF-κB-NLRP3 Pathway.

doi: 10.3389/fendo.2019.00778

Figure Lengend Snippet: FIGURE 3 | The NLRP3 inflammasome participates in excessive iodine-induced pyroptosis in TFCs. (A,B) The NLRP3 expression levels were measured by immunoblots when Nthy-ori 3-1 cells were treated with NaI with or without NAC (10 mM) or IKK-16 (2 µM) for 24 h. (C,D) Verification of the silencing efficiency of NLRP3 by siRNA in Nthy-ori3-1 cells is shown by immunoblots; NC indicates the negative control. (E,F) The protein levels of GSDMD were detected by immunoblots after transfection of siNLRP3 in NaI-treated Nthy-ori 3-1 cells. All statistical results shown are representative of three replicates. Significant differences and P-values were calculated by one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Thyroid sections were blocked with 2% bovine serum albumin in PBS for 30min and then incubated with mouse anti-human GSDMD or mouse antihuman IL-1β antibodies (Santa Cruz, NJ, USA) at 4◦C overnight.

Techniques: Expressing, Western Blot, Negative Control, Transfection

Journal: Frontiers in endocrinology

Article Title: Excessive Iodine Promotes Pyroptosis of Thyroid Follicular Epithelial Cells in Hashimoto's Thyroiditis Through the ROS-NF-κB-NLRP3 Pathway.

doi: 10.3389/fendo.2019.00778

Figure Lengend Snippet: FIGURE 5 | Proposed model of TFC pyroptosis in excessive iodine-promoted HT. Excessive iodine enters TFCs and induces the production of ROS, which activates NF-κB signaling and increases GSDMD-FL. In addition, the NLRP3 inflammasome is activated by NF-κB signaling, leading to GSDMD-FL cleavage into GSDMD-N, which triggers pore formation in the membrane and the release of mature IL-1β from cells, causing a sterile inflammatory response and further contributing to pyroptotic cell death and the subsequent promotion of HT.

Article Snippet: Thyroid sections were blocked with 2% bovine serum albumin in PBS for 30min and then incubated with mouse anti-human GSDMD or mouse antihuman IL-1β antibodies (Santa Cruz, NJ, USA) at 4◦C overnight.

Techniques: Membrane, Sterility

Journal: Kidney & blood pressure research

Article Title: Activation of TRPV1 Prevents Salt-Induced Kidney Damage and Hypertension After Renal Ischemia-Reperfusion Injury in Rats.

doi: 10.1159/000492412

Figure Lengend Snippet: Fig. 1. Effect of renal ischemia/reperfusion (I/R) and capsaicin pretreatment on the expression of renal TRPV1. Immunohistochemistry staining (a), representative Western blot (b) and quantification (c) of TRPV1 in renal tissue from rats in sham, I/R injury, I/R with pretreatment of a low dose of capsaicin (I/R+LCap), and I/R with pretreatment of a high dose of capsaicin (I/R+HCap) groups with HS diet for 4 weeks. Values are mean±SE (n=7 to 8). *P<0.05 compared with sham group; †P<0.05 compared with I/R group. Arrows indicate TRPV1 staining fibers. Scale bars, 50μm.

Article Snippet: USA), mouse anti-IL-1β antibody (1:200, sc-74138, Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-CTGF antibody (1:200, sc-25440), rabbit anti-collagen I antibody (1:200, sc-8784), or rabbit anti-collagen IV antibody (1:200, sc-11360), at 4°C overnight, followed by incubation with HRP-donkey anti-rabbit IgG (1:10, 000, 711-035-152, Jackson ImmunoResearch Laboratories, West Grove, PA, USA) or HRP-donkey anti-mouse IgG (1:10, 000, 711-035-151, Jackson ImmunoResearch Laboratories) for 2h at room temperature.

Techniques: Expressing, Immunohistochemistry, Staining, Western Blot

Journal: Kidney & blood pressure research

Article Title: Activation of TRPV1 Prevents Salt-Induced Kidney Damage and Hypertension After Renal Ischemia-Reperfusion Injury in Rats.

doi: 10.1159/000492412

Figure Lengend Snippet: Fig. 3. Effect of I/R, capsaicin pretreatment and high salt loading on renal function. 24-hour ratio of urine/ water intake m e a s u r e d once a week before and after I/R (a), and creatinine clearance (b) and plasma urea (c) and creatinine (d) measured at 1 day after I/R, 5-week after I/R, and 4-week after high salt loading in rats of sham, I/R injury, I/R+LCap, and I/R+HCap groups. Values are mean±SE (n=6-8). *P<0.05 compared with the sham group; †P<0.05 compared with the I/R group.

Article Snippet: USA), mouse anti-IL-1β antibody (1:200, sc-74138, Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-CTGF antibody (1:200, sc-25440), rabbit anti-collagen I antibody (1:200, sc-8784), or rabbit anti-collagen IV antibody (1:200, sc-11360), at 4°C overnight, followed by incubation with HRP-donkey anti-rabbit IgG (1:10, 000, 711-035-152, Jackson ImmunoResearch Laboratories, West Grove, PA, USA) or HRP-donkey anti-mouse IgG (1:10, 000, 711-035-151, Jackson ImmunoResearch Laboratories) for 2h at room temperature.

Techniques: Clinical Proteomics

Journal: Kidney & blood pressure research

Article Title: Activation of TRPV1 Prevents Salt-Induced Kidney Damage and Hypertension After Renal Ischemia-Reperfusion Injury in Rats.

doi: 10.1159/000492412

Figure Lengend Snippet: Fig. 4. Effect of I/R, capsaicin pretreatment and high salt loading on renal inflammation. Western blotting for the expression of TNF-α (a, b) and IL-1β (c, d) in the kidney from rats in sham, I/R injury, I/R+LCap, and I/R+HCap groups with HS diet for 4 weeks. Values are mean±SE (n=5). *P<0.05 compared with the sham group; †P<0.05 compared with the I/R group.

Article Snippet: USA), mouse anti-IL-1β antibody (1:200, sc-74138, Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-CTGF antibody (1:200, sc-25440), rabbit anti-collagen I antibody (1:200, sc-8784), or rabbit anti-collagen IV antibody (1:200, sc-11360), at 4°C overnight, followed by incubation with HRP-donkey anti-rabbit IgG (1:10, 000, 711-035-152, Jackson ImmunoResearch Laboratories, West Grove, PA, USA) or HRP-donkey anti-mouse IgG (1:10, 000, 711-035-151, Jackson ImmunoResearch Laboratories) for 2h at room temperature.

Techniques: Western Blot, Expressing

Journal: Glia

Article Title: The two pore potassium channel THIK ‐1 regulates NLRP3 inflammasome activation

doi: 10.1002/glia.24174

Figure Lengend Snippet: Pharmacological inhibition of two pore domain potassium channels blocks NLRP3 inflammasome activation and IL‐1β processing. (ai–iii) IL‐1β ELISA of the supernatant of pBMDMs primed with LPS (1 μg ml −1 , 4 h) followed by pretreatment with MCC950 (10 μM) or K + channel inhibitors TEA (50 mM), TRAM‐34 (10 μM), TPA (50 μM), ML‐133 (20 μM), quinine (100 μM), Guangitoxin‐1E (25 nM), PAP‐1 (2 μM), or Dofetilide (1 μM) for 15 min before stimulation with ATP (5 mM, 1 h) ( n = 4), silica (300 μg ml −1 , 4 h) ( n = 5) or imiquimod (75 μM, 2 h) ( n = 3). (Aiv) IL‐1β ELISA of the supernatant of pBMDMs primed with LPS (1 μg ml −1 , 4 h) followed by pretreatment with MCC950 (10 μM) or TEA (50 mM), TPA (50 μM), quinine (100 μM), for 15 min before stimulation with nigericin (10 μM, 1 h) ( n = 4). (b) Caspase‐1, IL‐ β and gasdermin D western blot of total cell lysates (cell lysate + supernatant) from LPS‐primed (1 μg ml −1 , 4 h) pBMDMs pretreated with vehicle control, TEA (50 mM), TPA (50 μM) or MCC950 (10 μM) for 15 min then stimulated with ATP (5 mM, 1 h). (c) IL‐1β ELISA of the supernatant of pBMDMs primed with LPS (1 μg ml −1 , 4 h) followed by pretreatment with MCC950 (10 μM) or TPA (3–300 μM) before stimulation with ATP (5 mM, 1 h) ( n = 5) or silica (300 μg ml −1 , 4 h) ( n = 3). (d) Caspase‐1 Glo assay to measure caspase‐1 activity of LPS‐primed (1 μg ml −1 , 4 h) pBMDMs pretreated with vehicle control, TEA (50 mM), TPA (50 μM) or MCC950 (10 μM) for 15 min then stimulated with ATP (5 mM, 1 h). **** p < .0001, *** p < .001, ** p < .01, * p < .05 determined by one‐way ANOVA with Dunnett's post hoc analysis. Values shown are the mean ± SEM

Article Snippet: Specific antibodies were used targeting: mouse IL‐1β (AF‐401, R&D), caspase‐1 p10 (EPR16883, Abcam), gasdermin D (ab209845, Abcam), NLRP3 (G‐20B‐0014‐C100, Adipogen), and β‐actin (Sigma).

Techniques: Inhibition, Activation Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Glo Assay, Activity Assay

Journal: Glia

Article Title: The two pore potassium channel THIK ‐1 regulates NLRP3 inflammasome activation

doi: 10.1002/glia.24174

Figure Lengend Snippet: Pharmacological inhibition of two pore potassium channels blocks priming of the NLRP3 inflammasome. (a) IL‐6 (i) and TNF (ii) ELISA and (b) LDH release assay of the supernatant of iBMDMs pretreated with Bay11(10 μM) or K + channel inhibitors TEA (50 mM), TRAM‐34 (10 μM), TPA (50 μM), ML‐133 (20 μM), quinine (100 μM), Guangitoxin‐1E (25 nM), PAP‐1 (2 μM) or Dofetilide (1 μM) for 15 min before priming with LPS (1 μg ml −1 , 4 h) ( n = 4). (c) NLRP3 and IL‐1β western blot of the supernatant and total cell lysates respectively of iBMDMs pretreated with TEA (50 mM), TPA (50 μM) or Bay11 (10 μM) for 15 min before priming with LPS (1 μg ml −1 , 4 h). **** p < .0001, ** p < .01 determined by one‐way ANOVA with Dunnett's post hoc analysis. Values shown are the mean ± SEM

Article Snippet: Specific antibodies were used targeting: mouse IL‐1β (AF‐401, R&D), caspase‐1 p10 (EPR16883, Abcam), gasdermin D (ab209845, Abcam), NLRP3 (G‐20B‐0014‐C100, Adipogen), and β‐actin (Sigma).

Techniques: Inhibition, Enzyme-linked Immunosorbent Assay, Lactate Dehydrogenase Assay, Western Blot

Journal: Glia

Article Title: The two pore potassium channel THIK ‐1 regulates NLRP3 inflammasome activation

doi: 10.1002/glia.24174

Figure Lengend Snippet: Potassium efflux is required for NLRP3 inflammasome activation but not ASC speck formation in response to ATP. (a, i) ASC speck formation measured in real time and (a, ii) ASC speck formation after 165 min of ATP stimulation from ASC‐mCherry iBMDMs primed with LPS (1 μg ml −1 , 4 h) followed by pretreatment vehicle control, TPA (50 μM) or MCC950 (10 μM) for 15 min before stimulation with ATP (5 mM) ( n = 6). (a, iii) ASC speck formation after 165 min from ASC‐mCherry iBMDMS primed with LPS (1 μg ml −1 , 4 h) followed by treatment with vehicle control, TPA (50 μM) or MCC950 (10 μM) in the absence of ATP ( n = 6). (b, i) IL‐1β ELISA of the supernatant of iBMDMs primed with LPS (1 μg ml −1 , 4 h) followed by incubation in a control (145 mM NaCl/ 5 mM KCl), high K + and normal Cl − (150 mM KCl), high K + and Cl − free (150 mM KGluconate) or control and MCC950 (10 μM) solution for 15 min before stimulation with ATP (5 mM, 1 h) ( n = 6). (b, ii) ASC speck formation measured in real time and (b, iii) ASC speck formation after 165 min of ATP stimulation from iBMDMs stably expressing ASC‐mCherry (ASC‐mCherry iBMDMs) primed with LPS (1 μg ml −1 , 4 h) followed by incubation in a control (145 mM NaCl/ 5 mM KCl), high K + and normal Cl − (150 mM KCl), high K + and Cl − free (150 mM KGluconate) or control and MCC950 (10 μM) solution for 15 min before stimulation with ATP (5 mM) ( n = 4). (b, iv) representative images of ASC‐mCherry iBMDMs after 165 min ATP stimulation (scale bar, 50 μm, arrows denote ASC specks). ASC speck experiments were performed in the presence of ac‐YVAD‐CMK (50 μM) to prevent pyroptosis and loss of ASC specks. **** p < .0001, * p < .05 determined by one‐way ANOVA with Dunnett's post hoc analysis. Values shown are the mean ± SEM

Article Snippet: Specific antibodies were used targeting: mouse IL‐1β (AF‐401, R&D), caspase‐1 p10 (EPR16883, Abcam), gasdermin D (ab209845, Abcam), NLRP3 (G‐20B‐0014‐C100, Adipogen), and β‐actin (Sigma).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Incubation, Stable Transfection, Expressing

Journal: Glia

Article Title: The two pore potassium channel THIK ‐1 regulates NLRP3 inflammasome activation

doi: 10.1002/glia.24174

Figure Lengend Snippet: Inhibition of THIK‐1 blocks NLRP3 activation in mixed glia and isolated microglia. (a) IL‐1β ELISA of the supernatant of primary mouse mixed glia primed with LPS (1 μg ml −1 , 4 h) followed by pretreatment with vehicle control, TPA (50 μM) or MCC950 (10 μM) for 15 min before stimulation with ATP (5 mM, 1 h) ( n = 5), silica (300 μg ml −1 ,4 h) ( n = 3), imiquimod (75 μM, 2 h) ( n = 4) or nigericin (10 μM, 1 h) ( n = 3). (b) IL‐1β ELISA of the supernatant of mouse primary microglia primed with LPS (1 μg ml −1 , 4 h) followed by pretreatment with vehicle control, TPA (50 μM) or MCC950 (10 μM) for 15 min before stimulation with ATP (5 mM, 1 h) or silica (300 μg ml −1 ,4 h) ( n = 3). **** p < .0001, *** p < .001, ** p < .01, * p < .05 determined by one‐way ANOVA with Dunnett's post hoc analysis. Values shown are the mean ± SEM

Article Snippet: Specific antibodies were used targeting: mouse IL‐1β (AF‐401, R&D), caspase‐1 p10 (EPR16883, Abcam), gasdermin D (ab209845, Abcam), NLRP3 (G‐20B‐0014‐C100, Adipogen), and β‐actin (Sigma).

Techniques: Inhibition, Activation Assay, Isolation, Enzyme-linked Immunosorbent Assay

Journal: Glia

Article Title: The two pore potassium channel THIK ‐1 regulates NLRP3 inflammasome activation

doi: 10.1002/glia.24174

Figure Lengend Snippet: THIK‐1 specifically regulates ATP‐induced NLRP3 activation in bone‐marrow‐derived macrophages. (a) IL‐1β ELISA of the supernatant of primary wild‐type (WT) and Kcnk13 knockout (KO) BMDMs primed with LPS (1 μg ml −1 , 4 h) followed by pretreatment with MCC950 (10 μM) for 15 min before stimulation with ATP (5 mM, 1 h) ( n = 7), silica (300 μg ml −1 ,4 h) ( n = 7), imiquimod (75 μM, 2 h) ( n = 4) or nigericin (10 μM, 1 h) ( n = 8). (b) Caspase‐1, IL‐1β and gasdermin D western blot of total cell lysates (cell lysate + supernatant) from LPS‐primed WT and Kcnk13 KO pBMDMs pretreated with vehicle control or MCC950 (10 μM) for 15 min before stimulated with ATP (5 mM, 1 h). (c and d) TNF and IL‐6 ELISA and NLRP3 and IL‐1β western blot of the supernatant and total cell lysates respectively of primary wild‐type (WT) and Kcnk13 knockout (KO) BMDMs pretreated with Bay11(10 μM) for 15 min before priming with LPS (1 μg ml −1 , 4 h) ( n = 7). **** p < .0001, *** p < .001, ** p < .01 determined by two‐way ANOVA with Bonferroni's post hoc analysis. Values shown are the mean ± SEM

Article Snippet: Specific antibodies were used targeting: mouse IL‐1β (AF‐401, R&D), caspase‐1 p10 (EPR16883, Abcam), gasdermin D (ab209845, Abcam), NLRP3 (G‐20B‐0014‐C100, Adipogen), and β‐actin (Sigma).

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

Journal: Glia

Article Title: The two pore potassium channel THIK ‐1 regulates NLRP3 inflammasome activation

doi: 10.1002/glia.24174

Figure Lengend Snippet: THIK‐1 regulates NLRP3 activation in primary adult microglia. (a) IL‐1β ELISA of the supernatant of primary wild‐type (WT) and Kcnk13 knockout (KO) adult microglia primed with LPS (1 μg ml −1 , 4 h) followed by pretreatment with MCC950 (10 μM) for 15 min before stimulation with ATP (5 mM, 1 h) ( n = 4), silica (300 μg ml −1 ,4 h) ( n = 3), imiquimod (75 μM, 2 h) ( n = 3) or nigericin (10 μM, 1 h) ( n = 4). (b) TNF ELISA of the supernatant of primary wild‐type (WT) and Kcnk13 knockout (KO) primary adult microglia pretreated with Bay11(10 μM) for 15 min before priming with LPS (1 μg ml −1 , 4 h) ( n = 5). **** p < .0001, *** p < .001, ** p < .01, * p < .05 determined by two‐way ANOVA with Bonferroni's post hoc analysis. Values shown are the mean ± SEM

Article Snippet: Specific antibodies were used targeting: mouse IL‐1β (AF‐401, R&D), caspase‐1 p10 (EPR16883, Abcam), gasdermin D (ab209845, Abcam), NLRP3 (G‐20B‐0014‐C100, Adipogen), and β‐actin (Sigma).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Knock-Out

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: AIM2 and NLRC4 inflammasomes contribute with ASC to acute brain injury independently of NLRP3

doi: 10.1073/pnas.1419090112

Figure Lengend Snippet: ASC-dependent mechanisms contribute to brain injury induced by cerebral ischemia. Infarct volume (A) as measured on cresyl violet-stained brain sections (B) is significantly reduced in ASC−/− mice (**P < 0.01 vs. WT, NLRP3−/− and NOD2−/−). (C) Average cerebral blood flow (CBF) values were unaltered during occlusion of the MCA (A–C; one-way ANOVA followed by Tukey’s post hoc test). (D) Neurological outcome was improved in ASC−/− mice (**P < 0.01, Kruskal–Wallis test followed by Dunn’s multiple comparison). (E) Schematic diagram summarizing the data. The dashed lines highlight dispensable pathways.

Article Snippet: In brief, appropriate mixtures of rat anti-mouse CD45 1:200 (Serotec), goat anti-mouse IL-1α, goat anti-mouse IL-1β 1:100 (R&D Systems), and rabbit anti-Iba1 1:1,000 (Wako Chemicals) antibodies were used followed by the appropriate fluorochrome-conjugated Alexa594 or Alexa488 donkey antisera (1:500, Invitrogen).

Techniques: Staining, Comparison

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: AIM2 and NLRC4 inflammasomes contribute with ASC to acute brain injury independently of NLRP3

doi: 10.1073/pnas.1419090112

Figure Lengend Snippet: ASC deficiency reduces inflammation in the brain independently of IL-1 production. Numbers of IL-1α– (A and B) and IL-1β– (C) positive microglia (A, Iba1+, arrowheads) are not significantly altered in the brain by NLRP3, NOD2, or ASC deficiency after cerebral ischemia. Schematic shows the location of IL-1–positive microglia in the ipsilateral hemisphere, which was similar in all animals. Numbers of activated microglia [D, expressing low levels of CD45 (CD45low), Iba1+], total microglial numbers (E), recruitment of leukocytes expressing high levels of CD45 (CD45high) (F), and vascular activation (G, blood vessels with high levels of tomato lectin staining, as shown on H in blue) have been assessed in the ipsilateral striatum and cerebral cortex. n = 6–9, two-way ANOVA followed by Tukey’s post hoc test. **P < 0.01, ***P < 0.001. (Scale bar, A, 50 μm; H, 100 μm.)

Article Snippet: In brief, appropriate mixtures of rat anti-mouse CD45 1:200 (Serotec), goat anti-mouse IL-1α, goat anti-mouse IL-1β 1:100 (R&D Systems), and rabbit anti-Iba1 1:1,000 (Wako Chemicals) antibodies were used followed by the appropriate fluorochrome-conjugated Alexa594 or Alexa488 donkey antisera (1:500, Invitrogen).

Techniques: Expressing, Activation Assay, Staining

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: AIM2 and NLRC4 inflammasomes contribute with ASC to acute brain injury independently of NLRP3

doi: 10.1073/pnas.1419090112

Figure Lengend Snippet: AIM2 and NLRC4 deficiency reduces inflammation in the brain independently of IL-1 production. Numbers of IL-1β– (A and B) positive microglia (arrowheads), IL-1α–positive microglia (C), activated microglia (A and D, arrowheads), and CD45high leukocytes (A and E, arrows) have been assessed in the ipsilateral hemisphere after MCAo (two-way ANOVA followed by Tukey’s post hoc test). (F) Protein levels of IL-1α and IL-1β were measured with cytometric bead array in liver and spleen homogenates (one-way ANOVA followed by Tukey’s post hoc test, **P < 0.01). n = 6–7. n.s., not significant. (Scale bar, 50 μm.)

Article Snippet: In brief, appropriate mixtures of rat anti-mouse CD45 1:200 (Serotec), goat anti-mouse IL-1α, goat anti-mouse IL-1β 1:100 (R&D Systems), and rabbit anti-Iba1 1:1,000 (Wako Chemicals) antibodies were used followed by the appropriate fluorochrome-conjugated Alexa594 or Alexa488 donkey antisera (1:500, Invitrogen).

Techniques: