Structured Review

InvivoGen ultrapure flagellin
Inhibitory effect of APO on ASC oligomerization. LPS-primed BMDMs were pretreated with APO or KCl (150 mM), and ASC oligomerization in the cells was determined by Western blotting upon stimulation with (a) 5 mM ATP for 1 h, (b) 150 μ g/mL silica for 3 h, (c) 10 μ M nigericin for 1 h, (d) 2 μ g/mL poly (dA:dT) for 1 h, and (e) 1.5 μ g/mL <t>flagellin</t> for 3 h. β -Actin is used as an internal control. (f) LPS-primed BMDMs were stimulated with 5 mM ATP for 1 h, 150 μ g/mL silica for 3 h, 10 μ M nigericin for 1 h, 2 μ g/mL poly (dA:dT) for 1 h, and 1.5 μ g/mL flagellin for 3 h and stained with anti-ASC antibody for ASC specks (red) and by DAPI for nuclei (blue). ASC specks were marked with arrows, and (g) cells with ASC specks are presented as a percentage of the positive cells. Data were expressed as the mean ± SEM ( n = 3). Statistical analysis was performed using Student's t -test. ∗ p
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1) Product Images from "Artemisia Extract Suppresses NLRP3 and AIM2 Inflammasome Activation by Inhibition of ASC Phosphorylation"

Article Title: Artemisia Extract Suppresses NLRP3 and AIM2 Inflammasome Activation by Inhibition of ASC Phosphorylation

Journal: Mediators of Inflammation

doi: 10.1155/2018/6054069

Inhibitory effect of APO on ASC oligomerization. LPS-primed BMDMs were pretreated with APO or KCl (150 mM), and ASC oligomerization in the cells was determined by Western blotting upon stimulation with (a) 5 mM ATP for 1 h, (b) 150 μ g/mL silica for 3 h, (c) 10 μ M nigericin for 1 h, (d) 2 μ g/mL poly (dA:dT) for 1 h, and (e) 1.5 μ g/mL flagellin for 3 h. β -Actin is used as an internal control. (f) LPS-primed BMDMs were stimulated with 5 mM ATP for 1 h, 150 μ g/mL silica for 3 h, 10 μ M nigericin for 1 h, 2 μ g/mL poly (dA:dT) for 1 h, and 1.5 μ g/mL flagellin for 3 h and stained with anti-ASC antibody for ASC specks (red) and by DAPI for nuclei (blue). ASC specks were marked with arrows, and (g) cells with ASC specks are presented as a percentage of the positive cells. Data were expressed as the mean ± SEM ( n = 3). Statistical analysis was performed using Student's t -test. ∗ p
Figure Legend Snippet: Inhibitory effect of APO on ASC oligomerization. LPS-primed BMDMs were pretreated with APO or KCl (150 mM), and ASC oligomerization in the cells was determined by Western blotting upon stimulation with (a) 5 mM ATP for 1 h, (b) 150 μ g/mL silica for 3 h, (c) 10 μ M nigericin for 1 h, (d) 2 μ g/mL poly (dA:dT) for 1 h, and (e) 1.5 μ g/mL flagellin for 3 h. β -Actin is used as an internal control. (f) LPS-primed BMDMs were stimulated with 5 mM ATP for 1 h, 150 μ g/mL silica for 3 h, 10 μ M nigericin for 1 h, 2 μ g/mL poly (dA:dT) for 1 h, and 1.5 μ g/mL flagellin for 3 h and stained with anti-ASC antibody for ASC specks (red) and by DAPI for nuclei (blue). ASC specks were marked with arrows, and (g) cells with ASC specks are presented as a percentage of the positive cells. Data were expressed as the mean ± SEM ( n = 3). Statistical analysis was performed using Student's t -test. ∗ p

Techniques Used: Western Blot, Staining

Effect of APO on inflammasome components. LPS-primed BMDMs (a–d, f) and nonprimed BMDMs (e, g) were pretreated with APO or zVAD (20 μ M) and stimulated with (a) 150 μ g/mL silica for 3 h, (b) 10 μ M nigericin for 1 h, (c) 5 mM ATP for 1 h, (d-e) 2 μ g/mL poly (dA:dT) for 1 h, and (f-g) 1.5 μ g/mL flagellin for 3 h. Mature IL-1 β and caspase-1 cleavage were measured in supernatant (Sup). NLRP3, pro-IL-1 β , procaspase-1, and ASC were measured in whole cell extract (WCE) by immunoblotting. β -Actin was used as an internal control. LPS, lipopolysaccharide; BMDMs, bone marrow-derived macrophages; APO, A. princeps extract; IL, interleukin; Nig, nigericin.
Figure Legend Snippet: Effect of APO on inflammasome components. LPS-primed BMDMs (a–d, f) and nonprimed BMDMs (e, g) were pretreated with APO or zVAD (20 μ M) and stimulated with (a) 150 μ g/mL silica for 3 h, (b) 10 μ M nigericin for 1 h, (c) 5 mM ATP for 1 h, (d-e) 2 μ g/mL poly (dA:dT) for 1 h, and (f-g) 1.5 μ g/mL flagellin for 3 h. Mature IL-1 β and caspase-1 cleavage were measured in supernatant (Sup). NLRP3, pro-IL-1 β , procaspase-1, and ASC were measured in whole cell extract (WCE) by immunoblotting. β -Actin was used as an internal control. LPS, lipopolysaccharide; BMDMs, bone marrow-derived macrophages; APO, A. princeps extract; IL, interleukin; Nig, nigericin.

Techniques Used: Derivative Assay

Effect of APO on cell viability and IL-1 β secretion in LPS-primed BMDMs. (a) BMDMs were treated with the indicated concentration of APO for 24 h. Cell viability was measured by the LDH assay. (b–f) LPS-primed BMDMs were pretreated with APO or zVAD (20 μ M), and IL-1 β secretion was determined by ELISA upon stimulation with (b) 150 μ g/mL silica for 3 h, (c) 10 μ M nigericin for 1 h, (d) 5 mM ATP for 1 h, (e) 2 μ g/mL poly (dA:dT) for 1 h, and (f) 1.5 μ g/mL flagellin for 3 h. (g) TNF- α release from LPS-primed BMDMs was determined upon stimulation with 10 μ M nigericin for 1 h. (h) The impact of APO on MSU-induced IL-1 β production in mice. Data were expressed as the mean ± SEM ( n = 3). Statistical analysis was performed using Student's t -test. ∗ p
Figure Legend Snippet: Effect of APO on cell viability and IL-1 β secretion in LPS-primed BMDMs. (a) BMDMs were treated with the indicated concentration of APO for 24 h. Cell viability was measured by the LDH assay. (b–f) LPS-primed BMDMs were pretreated with APO or zVAD (20 μ M), and IL-1 β secretion was determined by ELISA upon stimulation with (b) 150 μ g/mL silica for 3 h, (c) 10 μ M nigericin for 1 h, (d) 5 mM ATP for 1 h, (e) 2 μ g/mL poly (dA:dT) for 1 h, and (f) 1.5 μ g/mL flagellin for 3 h. (g) TNF- α release from LPS-primed BMDMs was determined upon stimulation with 10 μ M nigericin for 1 h. (h) The impact of APO on MSU-induced IL-1 β production in mice. Data were expressed as the mean ± SEM ( n = 3). Statistical analysis was performed using Student's t -test. ∗ p

Techniques Used: Concentration Assay, Lactate Dehydrogenase Assay, Enzyme-linked Immunosorbent Assay, Mouse Assay

2) Product Images from "Caspase-11 Mediates Neutrophil Chemotaxis and Extracellular Trap Formation During Acute Gouty Arthritis Through Alteration of Cofilin Phosphorylation"

Article Title: Caspase-11 Mediates Neutrophil Chemotaxis and Extracellular Trap Formation During Acute Gouty Arthritis Through Alteration of Cofilin Phosphorylation

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2019.02519

Caspase-11 −/− neutrophils produce less NETs independently of Rip3 phosphorylation and associated with altered cofilin phosphorylation in response to MSU in vitro . (A,B) Immunofluorescence assay of WT and caspase-11 −/− neutrophils treated with MSU, MSU + ATP, IL-1β, IL-1β + MSU, and PMA for 3 h. Cells were stained with neutrophil elastase (NE) and DAPI (DNA) to visualize NET formation, n = 3. (C) Quantification of NET formation in vitro . Response measured from WT and caspase-11 −/− neutrophils stimulated with MSU, ATP, IL-1β, combinations of treatments, and PMA. Cells were stained with Sytox green, n = 2 independent experiments (3 mice per group, total 6 mice), * p
Figure Legend Snippet: Caspase-11 −/− neutrophils produce less NETs independently of Rip3 phosphorylation and associated with altered cofilin phosphorylation in response to MSU in vitro . (A,B) Immunofluorescence assay of WT and caspase-11 −/− neutrophils treated with MSU, MSU + ATP, IL-1β, IL-1β + MSU, and PMA for 3 h. Cells were stained with neutrophil elastase (NE) and DAPI (DNA) to visualize NET formation, n = 3. (C) Quantification of NET formation in vitro . Response measured from WT and caspase-11 −/− neutrophils stimulated with MSU, ATP, IL-1β, combinations of treatments, and PMA. Cells were stained with Sytox green, n = 2 independent experiments (3 mice per group, total 6 mice), * p

Techniques Used: In Vitro, Immunofluorescence, Staining, Mouse Assay

3) Product Images from "Caspase-11 Mediates Neutrophil Chemotaxis and Extracellular Trap Formation During Acute Gouty Arthritis Through Alteration of Cofilin Phosphorylation"

Article Title: Caspase-11 Mediates Neutrophil Chemotaxis and Extracellular Trap Formation During Acute Gouty Arthritis Through Alteration of Cofilin Phosphorylation

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2019.02519

Caspase-11 −/− neutrophils produce less NETs independently of Rip3 phosphorylation and associated with altered cofilin phosphorylation in response to MSU in vitro . (A,B) Immunofluorescence assay of WT and caspase-11 −/− neutrophils treated with MSU, MSU + ATP, IL-1β, IL-1β + MSU, and PMA for 3 h. Cells were stained with neutrophil elastase (NE) and DAPI (DNA) to visualize NET formation, n = 3. (C) Quantification of NET formation in vitro . Response measured from WT and caspase-11 −/− neutrophils stimulated with MSU, ATP, IL-1β, combinations of treatments, and PMA. Cells were stained with Sytox green, n = 2 independent experiments (3 mice per group, total 6 mice), * p
Figure Legend Snippet: Caspase-11 −/− neutrophils produce less NETs independently of Rip3 phosphorylation and associated with altered cofilin phosphorylation in response to MSU in vitro . (A,B) Immunofluorescence assay of WT and caspase-11 −/− neutrophils treated with MSU, MSU + ATP, IL-1β, IL-1β + MSU, and PMA for 3 h. Cells were stained with neutrophil elastase (NE) and DAPI (DNA) to visualize NET formation, n = 3. (C) Quantification of NET formation in vitro . Response measured from WT and caspase-11 −/− neutrophils stimulated with MSU, ATP, IL-1β, combinations of treatments, and PMA. Cells were stained with Sytox green, n = 2 independent experiments (3 mice per group, total 6 mice), * p

Techniques Used: In Vitro, Immunofluorescence, Staining, Mouse Assay

4) Product Images from "The Immunomodulatory Metabolite Itaconate Modifies NLRP3 and Inhibits Inflammasome Activation"

Article Title: The Immunomodulatory Metabolite Itaconate Modifies NLRP3 and Inhibits Inflammasome Activation

Journal: Cell Metabolism

doi: 10.1016/j.cmet.2020.07.016

4-OI Reduces Inflammation in a Murine In Vivo Model of Peritonitis and Blocks NLRP3 Inflammasome Activation in Healthy Human and CAPS PBMCs (A–C) IL-1β concentration (A), IL-6 concentration (B), and neutrophil number (C) in the peritoneal lavage fluid of mice injected for 6 h with MSU crystals (30 mg/kg) ± 4-OI (50 mg/kg) (n = 3 for PBS groups, n = 8 for MSU groups). (D) LPS or Pam3CSK4 (14 h) and nigericin (2 h) induced IL-1β release (n = 5 for LPS + nigericin, n = 3 for Pam3CSK4 + nigericin) ± 4-OI or 4-O-2-MS (both 250 μM) from healthy human PBMCs. (E and F) Immunoblot analysis (E) and quantification by densitometry (F, n = 3) of pro- and mature IL-1β protein in lysates and supernatants of human PBMCs treated with LPS (14 h) and nigericin (2 h) ± 4-OI (250 μM). (G) LPS (1 h) induced IL-1β release (n = 3) ± 4-OI (250 μM) or MCC950 (500 nM) from PBMCs isolated from CAPS patients. ∗ p
Figure Legend Snippet: 4-OI Reduces Inflammation in a Murine In Vivo Model of Peritonitis and Blocks NLRP3 Inflammasome Activation in Healthy Human and CAPS PBMCs (A–C) IL-1β concentration (A), IL-6 concentration (B), and neutrophil number (C) in the peritoneal lavage fluid of mice injected for 6 h with MSU crystals (30 mg/kg) ± 4-OI (50 mg/kg) (n = 3 for PBS groups, n = 8 for MSU groups). (D) LPS or Pam3CSK4 (14 h) and nigericin (2 h) induced IL-1β release (n = 5 for LPS + nigericin, n = 3 for Pam3CSK4 + nigericin) ± 4-OI or 4-O-2-MS (both 250 μM) from healthy human PBMCs. (E and F) Immunoblot analysis (E) and quantification by densitometry (F, n = 3) of pro- and mature IL-1β protein in lysates and supernatants of human PBMCs treated with LPS (14 h) and nigericin (2 h) ± 4-OI (250 μM). (G) LPS (1 h) induced IL-1β release (n = 3) ± 4-OI (250 μM) or MCC950 (500 nM) from PBMCs isolated from CAPS patients. ∗ p

Techniques Used: In Vivo, Activation Assay, Concentration Assay, Mouse Assay, Injection, Isolation

5) Product Images from "MicroRNA-488 and -920 regulate the production of proinflammatory cytokines in acute gouty arthritis"

Article Title: MicroRNA-488 and -920 regulate the production of proinflammatory cytokines in acute gouty arthritis

Journal: Arthritis Research & Therapy

doi: 10.1186/s13075-017-1418-6

MicroRNA (miR, miRNA)-488 and miR-920 suppress monosodium urate (MSU)-induced expression of proinflammatory cytokines in THP-1 cells. The miRNA mimics or negative control (NC) mimics (50 nM) were transfected into THP-1 cells using Lipofectamine RNAiMAX reagent in accordance with the manufacturer’s instructions. After 24 h of transfection, cells were stimulated for 3 h with 0.5 μM 12-myristate 13-acetate. Then, cells were washed and stimulated with 250 μg/ml MSU crystals for 24 h to detect the production of proinflammatory cytokines. After the treatment, the cells were collected and analyzed by quantitative real-time polymerase chain reaction ( a , c ). The cell culture supernatants were also collected to detect the concentrations of interleukin (IL)-8 and tumor necrosis factor (TNF)-α by enzyme-linked immunosorbent assay ( b , d ). Values are expressed as mean ± SEM of three independent experiments, each of which was run in triplicate. # P
Figure Legend Snippet: MicroRNA (miR, miRNA)-488 and miR-920 suppress monosodium urate (MSU)-induced expression of proinflammatory cytokines in THP-1 cells. The miRNA mimics or negative control (NC) mimics (50 nM) were transfected into THP-1 cells using Lipofectamine RNAiMAX reagent in accordance with the manufacturer’s instructions. After 24 h of transfection, cells were stimulated for 3 h with 0.5 μM 12-myristate 13-acetate. Then, cells were washed and stimulated with 250 μg/ml MSU crystals for 24 h to detect the production of proinflammatory cytokines. After the treatment, the cells were collected and analyzed by quantitative real-time polymerase chain reaction ( a , c ). The cell culture supernatants were also collected to detect the concentrations of interleukin (IL)-8 and tumor necrosis factor (TNF)-α by enzyme-linked immunosorbent assay ( b , d ). Values are expressed as mean ± SEM of three independent experiments, each of which was run in triplicate. # P

Techniques Used: Expressing, Negative Control, Transfection, Real-time Polymerase Chain Reaction, Cell Culture, Enzyme-linked Immunosorbent Assay

Monosodium urate (MSU) crystals promote the expression of proinflammatory cytokines in THP-1 cells. THP-1 cells were stimulated by the indicated concentration of MSU crystals. The messenger (mRNA) expression of interleukin (IL)-1β, IL-8, and tumor necrosis factor (TNF)-α were detected by quantitative real-time polymerase chain reaction ( a – c ). Protein expression of IL-1β, IL-8, and TNF-α was detected by enzyme-linked immunosorbent assay ( d – f ). Values are expressed as mean ± SEM of three independent experiments, each of which was run in triplicate. * P
Figure Legend Snippet: Monosodium urate (MSU) crystals promote the expression of proinflammatory cytokines in THP-1 cells. THP-1 cells were stimulated by the indicated concentration of MSU crystals. The messenger (mRNA) expression of interleukin (IL)-1β, IL-8, and tumor necrosis factor (TNF)-α were detected by quantitative real-time polymerase chain reaction ( a – c ). Protein expression of IL-1β, IL-8, and TNF-α was detected by enzyme-linked immunosorbent assay ( d – f ). Values are expressed as mean ± SEM of three independent experiments, each of which was run in triplicate. * P

Techniques Used: Expressing, Concentration Assay, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

6) Product Images from "A synthetic cationic antimicrobial peptide inhibits inflammatory response and the NLRP3 inflammasome by neutralizing LPS and ATP"

Article Title: A synthetic cationic antimicrobial peptide inhibits inflammatory response and the NLRP3 inflammasome by neutralizing LPS and ATP

Journal: PLoS ONE

doi: 10.1371/journal.pone.0182057

Effects of GW-A2 on NLRP3 inflammasome activation. ( A and B ) J774A.1 macrophages were incubated for 30 min with or without 2 μM of GW-A2, for 5.5 h with or without 0.1 μg/ml of E . coli LPS, and then for 30 min with or without 5 mM of ATP ( A ) or 10 μM of nigericin ( B ). The levels of IL-1β in the culture medium and activated caspase-1 (p10) in the cells were measured by ELISA and Western blotting, respectively. ( C — F ) J774A.1 macrophages were incubated for 5.5 h with 0.1 μg/ml of E . coli LPS, for 30 min with or without 2 μM of GW-A2, and then for 30 min with or without 5 mM of ATP ( C and E ), 10 μM of nigericin ( D ) or 100 μg/ml of MSU ( F ). The levels of IL-1β and TNF-α in the culture medium and activated caspase-1 (p10) in the cells were measured by ELISA and Western blotting, respectively. The data are expressed as the mean ± SD of three independent experiments. The Western blotting results shown are a representative experiment of three independent experiments. *, ** and *** indicate significant differences, representing p
Figure Legend Snippet: Effects of GW-A2 on NLRP3 inflammasome activation. ( A and B ) J774A.1 macrophages were incubated for 30 min with or without 2 μM of GW-A2, for 5.5 h with or without 0.1 μg/ml of E . coli LPS, and then for 30 min with or without 5 mM of ATP ( A ) or 10 μM of nigericin ( B ). The levels of IL-1β in the culture medium and activated caspase-1 (p10) in the cells were measured by ELISA and Western blotting, respectively. ( C — F ) J774A.1 macrophages were incubated for 5.5 h with 0.1 μg/ml of E . coli LPS, for 30 min with or without 2 μM of GW-A2, and then for 30 min with or without 5 mM of ATP ( C and E ), 10 μM of nigericin ( D ) or 100 μg/ml of MSU ( F ). The levels of IL-1β and TNF-α in the culture medium and activated caspase-1 (p10) in the cells were measured by ELISA and Western blotting, respectively. The data are expressed as the mean ± SD of three independent experiments. The Western blotting results shown are a representative experiment of three independent experiments. *, ** and *** indicate significant differences, representing p

Techniques Used: Activation Assay, Incubation, Enzyme-linked Immunosorbent Assay, Western Blot

7) Product Images from "Uric acid crystals induces placental inflammation and alters trophoblast function via an IL-1-dependent pathway: implication for FGR"

Article Title: Uric acid crystals induces placental inflammation and alters trophoblast function via an IL-1-dependent pathway: implication for FGR

Journal: Journal of immunology (Baltimore, Md. : 1950)

doi: 10.4049/jimmunol.1601179

Histological analysis of IL-1β staining in the placenta following MSU crystals treatment.
Figure Legend Snippet: Histological analysis of IL-1β staining in the placenta following MSU crystals treatment.

Techniques Used: Staining

8) Product Images from "Uric acid crystals induces placental inflammation and alters trophoblast function via an IL-1-dependent pathway: implication for FGR"

Article Title: Uric acid crystals induces placental inflammation and alters trophoblast function via an IL-1-dependent pathway: implication for FGR

Journal: Journal of immunology (Baltimore, Md. : 1950)

doi: 10.4049/jimmunol.1601179

Histological analysis of IL-1β staining in the placenta following MSU crystals treatment.
Figure Legend Snippet: Histological analysis of IL-1β staining in the placenta following MSU crystals treatment.

Techniques Used: Staining

9) Product Images from "The PYRIN domain-only protein POP3 inhibits AIM2-like receptor inflammasomes and regulates responses to DNA virus infections"

Article Title: The PYRIN domain-only protein POP3 inhibits AIM2-like receptor inflammasomes and regulates responses to DNA virus infections

Journal: Nature immunology

doi: 10.1038/ni.2829

POP3 expression in BMDM inhibits AIM2 and IFI16 inflammasome-mediated cytokine release (a–h) BMDM were infected with MVA, MCMV or KSHV, treated with LPS/ATP, MDP, MSU or transfected with poly(dA:dT) or flagellin for 16 h and analyzed for a–c, g, mature IL-1β, d, mature IL-18, e, IL-6 and f, TNF, h, IFN-β by ELISA, as indicated (n = 3 [a, b, d–h], n = 6 [c] ± s.e.m.). Data are representative of three (a–g), two (c, h) experiments. (a) * P =0.0064, ** P =0.0188; (b) * P =0.0015, ** P =0.0026; (c) * P =0.0020; (d) * P
Figure Legend Snippet: POP3 expression in BMDM inhibits AIM2 and IFI16 inflammasome-mediated cytokine release (a–h) BMDM were infected with MVA, MCMV or KSHV, treated with LPS/ATP, MDP, MSU or transfected with poly(dA:dT) or flagellin for 16 h and analyzed for a–c, g, mature IL-1β, d, mature IL-18, e, IL-6 and f, TNF, h, IFN-β by ELISA, as indicated (n = 3 [a, b, d–h], n = 6 [c] ± s.e.m.). Data are representative of three (a–g), two (c, h) experiments. (a) * P =0.0064, ** P =0.0188; (b) * P =0.0015, ** P =0.0026; (c) * P =0.0020; (d) * P

Techniques Used: Expressing, Infection, Transfection, Enzyme-linked Immunosorbent Assay

Silencing of POP3 in hMΦ enhances ALR-mediated IL-1β and IL-18 release (a–f, j, k) hMΦ were transfected with either control or POP3 siRNAs, confirmed for POP3 silencing by RT-PCR ( a, insert), and treated with LPS, MDP, MSU or SiO 2 , transfected with poly(dA:dT), LeTx, or flagellin, or infected with MVA as indicated for 16 h and analyzed a–c, for mature IL-1β; d, mature IL-18; e, TNF and k, IFN-β by ELISA (n = 3 ± s.e.m.). (f) POP3 silencing and ASC , AIM2 and IFI16 expression were determined by Real-Time PCR. (g–i) Stable THP-1 (GFP) or THP-1 (GFP-POP3) cells were analyzed for secretion of g, IL-1β and h, IL-18 in response to MVA and MCMV infection, transfection of poly(dA:dT) and treatment with MSU by ELISA (n = 3 ± s.e.m.). i, THP-1 (GFP) or THP-1 (GFP-POP3) cells were treated with IFN-β for the indicated times and expression of GFP and POP3 was analyzed by immunoblot and compared to β-tubulin. (j, k) hMΦ were transfected with siRNAs as above, j, transduced with either control GFP or GFP-POP3 expressing AdV, and either mock or MVA infected and analyzed for mature IL-1β by ELISA (n = 3 ± s.e.m.) or k, analyzed for secretion of IFN-β by ELISA (n = 3 ± s.e.m.). Data are representative of 3 experiments (a–f) and 2 experiments (g, h, j, k) or one experiment (i). (a) * P
Figure Legend Snippet: Silencing of POP3 in hMΦ enhances ALR-mediated IL-1β and IL-18 release (a–f, j, k) hMΦ were transfected with either control or POP3 siRNAs, confirmed for POP3 silencing by RT-PCR ( a, insert), and treated with LPS, MDP, MSU or SiO 2 , transfected with poly(dA:dT), LeTx, or flagellin, or infected with MVA as indicated for 16 h and analyzed a–c, for mature IL-1β; d, mature IL-18; e, TNF and k, IFN-β by ELISA (n = 3 ± s.e.m.). (f) POP3 silencing and ASC , AIM2 and IFI16 expression were determined by Real-Time PCR. (g–i) Stable THP-1 (GFP) or THP-1 (GFP-POP3) cells were analyzed for secretion of g, IL-1β and h, IL-18 in response to MVA and MCMV infection, transfection of poly(dA:dT) and treatment with MSU by ELISA (n = 3 ± s.e.m.). i, THP-1 (GFP) or THP-1 (GFP-POP3) cells were treated with IFN-β for the indicated times and expression of GFP and POP3 was analyzed by immunoblot and compared to β-tubulin. (j, k) hMΦ were transfected with siRNAs as above, j, transduced with either control GFP or GFP-POP3 expressing AdV, and either mock or MVA infected and analyzed for mature IL-1β by ELISA (n = 3 ± s.e.m.) or k, analyzed for secretion of IFN-β by ELISA (n = 3 ± s.e.m.). Data are representative of 3 experiments (a–f) and 2 experiments (g, h, j, k) or one experiment (i). (a) * P

Techniques Used: Transfection, Reverse Transcription Polymerase Chain Reaction, Infection, Enzyme-linked Immunosorbent Assay, Expressing, Real-time Polymerase Chain Reaction, Transduction

10) Product Images from "NLRP3 tyrosine phosphorylation is controlled by protein tyrosine phosphatase PTPN22"

Article Title: NLRP3 tyrosine phosphorylation is controlled by protein tyrosine phosphatase PTPN22

Journal: The Journal of Clinical Investigation

doi: 10.1172/JCI83669

NLRP3 is tyrosine phosphorylated. THP-1 and MM6 cells ( A and C ), BMDCs ( B ), and PBMCs ( D ) were treated with upLPS for 12 hours prior to activation with MDP (100 ng/ml, 24 hours), MSU (100 ng/ml, 6 hours), TiO 2 (150 ng/ml, 24 hours), SiO 2 (150 ng/ml, 24 hours), ATP (2 mM, 30 minutes), dsDNA, or flagellin, as indicated. NLRP3 was precipitated from cell lysates, and precipitates were analyzed for tyrosine phosphorylation, coprecipitated PTPN22, or coprecipitated PTPN2. ( D ) PTPN22 was precipitated in addition to NLRP3 and analyzed for coprecipitated NLRP3. ( E ) Amounts of NLRP3 and pTyr in NLRP3 precipitates were quantified using standard curves for NLRP3 and a pTyr peptide. The graph below the representative Western blots shows statistical analysis of densitometry. ND, not detected. Left and right blots in E were run on the same gel, but were discontinuous. Numbers below the blots in D show the amount of loaded NLRP3 or pTyr peptide (left) and measured amounts (right), respectively. The pound symbol (#) marks blots where the 130-kDa and 120-kDa forms of NLRP3 are not completely separated and appear as single bands. Data shown are from 1 of 3–5 independent experiments. * P
Figure Legend Snippet: NLRP3 is tyrosine phosphorylated. THP-1 and MM6 cells ( A and C ), BMDCs ( B ), and PBMCs ( D ) were treated with upLPS for 12 hours prior to activation with MDP (100 ng/ml, 24 hours), MSU (100 ng/ml, 6 hours), TiO 2 (150 ng/ml, 24 hours), SiO 2 (150 ng/ml, 24 hours), ATP (2 mM, 30 minutes), dsDNA, or flagellin, as indicated. NLRP3 was precipitated from cell lysates, and precipitates were analyzed for tyrosine phosphorylation, coprecipitated PTPN22, or coprecipitated PTPN2. ( D ) PTPN22 was precipitated in addition to NLRP3 and analyzed for coprecipitated NLRP3. ( E ) Amounts of NLRP3 and pTyr in NLRP3 precipitates were quantified using standard curves for NLRP3 and a pTyr peptide. The graph below the representative Western blots shows statistical analysis of densitometry. ND, not detected. Left and right blots in E were run on the same gel, but were discontinuous. Numbers below the blots in D show the amount of loaded NLRP3 or pTyr peptide (left) and measured amounts (right), respectively. The pound symbol (#) marks blots where the 130-kDa and 120-kDa forms of NLRP3 are not completely separated and appear as single bands. Data shown are from 1 of 3–5 independent experiments. * P

Techniques Used: Activation Assay, Western Blot

PTPN22 interacts with NLRP3 upon inflammasome activation. THP-1 cells, MM6 cells, and BMDCs were treated with upLPS for 12 hours prior to activation with MDP (100 ng/ml, 24 hours), MSU (100 ng/ml, 6 hours), TiO 2 (150 ng/ml, 24 hours), SiO 2 (150 ng/ml, 24 hours), ATP (2 mM, 30 minutes), dsDNA, or flagellin, as indicated. ( A and C ) PTPN22 was precipitated from THP-1 or MM6 lysates and analyzed by Western blot for coprecipitated NLRP3. ( B ) PTPN22 was precipitated from BMDCs and analyzed for NLRP3. ( D ) PTPN22 was precipitated from dsDNA-treated THP-1 cells or BMDCs and analyzed by Western blot for coprecipitation of AIM2. ( E ) PTPN22 was precipitated from dsDNA-treated THP-1 cells or BMDCs and analyzed by Western blot for coprecipitation of NLRC4. Data are representative of 1 of 3–5 independent experiments.
Figure Legend Snippet: PTPN22 interacts with NLRP3 upon inflammasome activation. THP-1 cells, MM6 cells, and BMDCs were treated with upLPS for 12 hours prior to activation with MDP (100 ng/ml, 24 hours), MSU (100 ng/ml, 6 hours), TiO 2 (150 ng/ml, 24 hours), SiO 2 (150 ng/ml, 24 hours), ATP (2 mM, 30 minutes), dsDNA, or flagellin, as indicated. ( A and C ) PTPN22 was precipitated from THP-1 or MM6 lysates and analyzed by Western blot for coprecipitated NLRP3. ( B ) PTPN22 was precipitated from BMDCs and analyzed for NLRP3. ( D ) PTPN22 was precipitated from dsDNA-treated THP-1 cells or BMDCs and analyzed by Western blot for coprecipitation of AIM2. ( E ) PTPN22 was precipitated from dsDNA-treated THP-1 cells or BMDCs and analyzed by Western blot for coprecipitation of NLRC4. Data are representative of 1 of 3–5 independent experiments.

Techniques Used: Activation Assay, Western Blot

Knockdown of PTPN22 reduces IL-1β secretion. PTPN22 knockdown was induced in THP-1 cells using lentiviral shRNA expression vectors. Cells were pretreated for 12 hours with upLPS before activation with MDP (100 ng/ml, 24 hours), MSU (150 ng/ml, 6 hours), TiO 2 (150 ng/ml, 24 hours), SiO 2 (150 ng/ml, 12 hours), or ATP (200 mM, 30 minutes). ( A ) Cell culture supernatants were analyzed for IL-1β secretion, LDH release, and IL-6 secretion by ELISA. ( B ) Cell lysates or supernatants were analyzed for caspase-1 (Casp-1), caspase-3 (Casp-3), IL-1β, NLRP3, ASC, and PTPN22 expression. Blots for IL-1β were run on the same gel but were discontinuous. Data are representative of 1 of at least 3 independent experiments with 3–5 replicas ( n = 3–5; * P
Figure Legend Snippet: Knockdown of PTPN22 reduces IL-1β secretion. PTPN22 knockdown was induced in THP-1 cells using lentiviral shRNA expression vectors. Cells were pretreated for 12 hours with upLPS before activation with MDP (100 ng/ml, 24 hours), MSU (150 ng/ml, 6 hours), TiO 2 (150 ng/ml, 24 hours), SiO 2 (150 ng/ml, 12 hours), or ATP (200 mM, 30 minutes). ( A ) Cell culture supernatants were analyzed for IL-1β secretion, LDH release, and IL-6 secretion by ELISA. ( B ) Cell lysates or supernatants were analyzed for caspase-1 (Casp-1), caspase-3 (Casp-3), IL-1β, NLRP3, ASC, and PTPN22 expression. Blots for IL-1β were run on the same gel but were discontinuous. Data are representative of 1 of at least 3 independent experiments with 3–5 replicas ( n = 3–5; * P

Techniques Used: shRNA, Expressing, Activation Assay, Cell Culture, Enzyme-linked Immunosorbent Assay

11) Product Images from "NLRP3-Inflammasome Activating DAMPs Stimulate an Inflammatory Response in Glia in the Absence of Priming Which Contributes to Brain Inflammation after Injury"

Article Title: NLRP3-Inflammasome Activating DAMPs Stimulate an Inflammatory Response in Glia in the Absence of Priming Which Contributes to Brain Inflammation after Injury

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2012.00288

DAMPs induce the release of proteases from cultured mixed glia . Cultured mixed glia were treated with the NLRP3-activating DAMPs ATP (5 mM), MSU, and CPPD (both 250 μg/ml) or the PAMP LPS (1 μg/ml) for 24 h. Supernatants were analyzed for the release of mature Cathepsin B (28–30 kDa) by Western blot (A) . To measure the release of martix metalloproteases by zymography all samples were loaded neat onto gels except for one of the ATP-treated supernatants (+*) which was diluted 1:3 (B) . Blots and gels shown are representative of at least five separate experiments.
Figure Legend Snippet: DAMPs induce the release of proteases from cultured mixed glia . Cultured mixed glia were treated with the NLRP3-activating DAMPs ATP (5 mM), MSU, and CPPD (both 250 μg/ml) or the PAMP LPS (1 μg/ml) for 24 h. Supernatants were analyzed for the release of mature Cathepsin B (28–30 kDa) by Western blot (A) . To measure the release of martix metalloproteases by zymography all samples were loaded neat onto gels except for one of the ATP-treated supernatants (+*) which was diluted 1:3 (B) . Blots and gels shown are representative of at least five separate experiments.

Techniques Used: Cell Culture, Western Blot, Zymography

Effects of DAMPs on pro-inflammatory protein levels in cultured mixed glia . Protein levels of pro-inflammatory mediators were measured by specific ELISAs after 24 h (A–D) or 4 h (E) exposure to the NLRP3-activating DAMPs ATP (5 mM), MSU, and CPPD [both 250 μg/ml; (Ai) ] or the PAMP LPS [1 μg/ml; (Aii) ]. The proteins analyzed were IL-1β (A) , IL-1α (B) , IL-6 (C,E) , CXCL1 (D,E) . Data are pooled samples from at least five separate experiments. *** P
Figure Legend Snippet: Effects of DAMPs on pro-inflammatory protein levels in cultured mixed glia . Protein levels of pro-inflammatory mediators were measured by specific ELISAs after 24 h (A–D) or 4 h (E) exposure to the NLRP3-activating DAMPs ATP (5 mM), MSU, and CPPD [both 250 μg/ml; (Ai) ] or the PAMP LPS [1 μg/ml; (Aii) ]. The proteins analyzed were IL-1β (A) , IL-1α (B) , IL-6 (C,E) , CXCL1 (D,E) . Data are pooled samples from at least five separate experiments. *** P

Techniques Used: Cell Culture

Effects of DAMPs on surface marker expression in microglia cells . The expression of surface markers of microglia activation MHC class II (A) , and CD11c (B) were measured after 24 h exposure to the NLRP3-activating DAMPs ATP (5 mM), MSU, and CPPD [both 250 μg/ml; (Bi) ] or the PAMP LPS [1 μg/ml; (Bii) ]. Flow cytometry was used to quantify surface marker expression. Data are expressed as a percentage of cells that co-express CD45 and CD11b. Representative dot plots showing the CD45 and CD11b expressing cell population plus and minus LPS treatment are also shown (Biii) . Data are pooled samples from at least five separate experiments. *** P
Figure Legend Snippet: Effects of DAMPs on surface marker expression in microglia cells . The expression of surface markers of microglia activation MHC class II (A) , and CD11c (B) were measured after 24 h exposure to the NLRP3-activating DAMPs ATP (5 mM), MSU, and CPPD [both 250 μg/ml; (Bi) ] or the PAMP LPS [1 μg/ml; (Bii) ]. Flow cytometry was used to quantify surface marker expression. Data are expressed as a percentage of cells that co-express CD45 and CD11b. Representative dot plots showing the CD45 and CD11b expressing cell population plus and minus LPS treatment are also shown (Biii) . Data are pooled samples from at least five separate experiments. *** P

Techniques Used: Marker, Expressing, Activation Assay, Flow Cytometry, Cytometry

The effects of PAMPs and DAMPs on the release of IL-1 from cultured mixed glia . Cultured mixed glia were treated (1 h) with the NLRP3-activating DAMPs ATP (5 mM), MSU, and CPPD (both 250 μg/ml) plus and minus a 24 h priming stimulus with the PAMP LPS (1 μg/ml). Release of both IL-1β (A) and IL-1α (B) were quantified by ELISA (Ai) , and processing of pro- (31 kDa) to mature (17 kDa) IL-1β in PAMP and DAMP treated cells was analyzed by Western blot (Aii) . Data are pooled samples from at least five separate experiments. ** P
Figure Legend Snippet: The effects of PAMPs and DAMPs on the release of IL-1 from cultured mixed glia . Cultured mixed glia were treated (1 h) with the NLRP3-activating DAMPs ATP (5 mM), MSU, and CPPD (both 250 μg/ml) plus and minus a 24 h priming stimulus with the PAMP LPS (1 μg/ml). Release of both IL-1β (A) and IL-1α (B) were quantified by ELISA (Ai) , and processing of pro- (31 kDa) to mature (17 kDa) IL-1β in PAMP and DAMP treated cells was analyzed by Western blot (Aii) . Data are pooled samples from at least five separate experiments. ** P

Techniques Used: Cell Culture, Enzyme-linked Immunosorbent Assay, Western Blot

Effects of DAMPs on the expression of pro-inflammatory genes in cultured mixed glia . mRNA levels of pro-inflammatory genes were measured by qPCR after 4 h exposure to the NLRP3-activating DAMPs ATP (5 mM), MSU, and CPPD (both 250 μg/ml) (Ai) or the PAMP LPS (1 μg/ml) (Aii) . Data were normalized to expression levels of the housekeeping gene SDHA across each treatment and fold change was expressed relative to basal RNA levels from untreated mixed glia. The genes analyzed were IL-1β (A) , IL-1α (B) , caspase-1 (C) , NLRP3 (D) , ASC (E) , iNOS (F) , IL-6 (G) , TNFα (H) , and CXCL1 (I) . Data are pooled samples from at least five separate experiments. ** P
Figure Legend Snippet: Effects of DAMPs on the expression of pro-inflammatory genes in cultured mixed glia . mRNA levels of pro-inflammatory genes were measured by qPCR after 4 h exposure to the NLRP3-activating DAMPs ATP (5 mM), MSU, and CPPD (both 250 μg/ml) (Ai) or the PAMP LPS (1 μg/ml) (Aii) . Data were normalized to expression levels of the housekeeping gene SDHA across each treatment and fold change was expressed relative to basal RNA levels from untreated mixed glia. The genes analyzed were IL-1β (A) , IL-1α (B) , caspase-1 (C) , NLRP3 (D) , ASC (E) , iNOS (F) , IL-6 (G) , TNFα (H) , and CXCL1 (I) . Data are pooled samples from at least five separate experiments. ** P

Techniques Used: Expressing, Cell Culture, Real-time Polymerase Chain Reaction

12) Product Images from "NLRP3-Inflammasome Activating DAMPs Stimulate an Inflammatory Response in Glia in the Absence of Priming Which Contributes to Brain Inflammation after Injury"

Article Title: NLRP3-Inflammasome Activating DAMPs Stimulate an Inflammatory Response in Glia in the Absence of Priming Which Contributes to Brain Inflammation after Injury

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2012.00288

DAMPs induce the release of proteases from cultured mixed glia . Cultured mixed glia were treated with the NLRP3-activating DAMPs ATP (5 mM), MSU, and CPPD (both 250 μg/ml) or the PAMP LPS (1 μg/ml) for 24 h. Supernatants were analyzed for the release of mature Cathepsin B (28–30 kDa) by Western blot (A) . To measure the release of martix metalloproteases by zymography all samples were loaded neat onto gels except for one of the ATP-treated supernatants (+*) which was diluted 1:3 (B) . Blots and gels shown are representative of at least five separate experiments.
Figure Legend Snippet: DAMPs induce the release of proteases from cultured mixed glia . Cultured mixed glia were treated with the NLRP3-activating DAMPs ATP (5 mM), MSU, and CPPD (both 250 μg/ml) or the PAMP LPS (1 μg/ml) for 24 h. Supernatants were analyzed for the release of mature Cathepsin B (28–30 kDa) by Western blot (A) . To measure the release of martix metalloproteases by zymography all samples were loaded neat onto gels except for one of the ATP-treated supernatants (+*) which was diluted 1:3 (B) . Blots and gels shown are representative of at least five separate experiments.

Techniques Used: Cell Culture, Western Blot, Zymography

Effects of DAMPs on pro-inflammatory protein levels in cultured mixed glia . Protein levels of pro-inflammatory mediators were measured by specific ELISAs after 24 h (A–D) or 4 h (E) exposure to the NLRP3-activating DAMPs ATP (5 mM), MSU, and CPPD [both 250 μg/ml; (Ai) ] or the PAMP LPS [1 μg/ml; (Aii) ]. The proteins analyzed were IL-1β (A) , IL-1α (B) , IL-6 (C,E) , CXCL1 (D,E) . Data are pooled samples from at least five separate experiments. *** P
Figure Legend Snippet: Effects of DAMPs on pro-inflammatory protein levels in cultured mixed glia . Protein levels of pro-inflammatory mediators were measured by specific ELISAs after 24 h (A–D) or 4 h (E) exposure to the NLRP3-activating DAMPs ATP (5 mM), MSU, and CPPD [both 250 μg/ml; (Ai) ] or the PAMP LPS [1 μg/ml; (Aii) ]. The proteins analyzed were IL-1β (A) , IL-1α (B) , IL-6 (C,E) , CXCL1 (D,E) . Data are pooled samples from at least five separate experiments. *** P

Techniques Used: Cell Culture

Effects of DAMPs on surface marker expression in microglia cells . The expression of surface markers of microglia activation MHC class II (A) , and CD11c (B) were measured after 24 h exposure to the NLRP3-activating DAMPs ATP (5 mM), MSU, and CPPD [both 250 μg/ml; (Bi) ] or the PAMP LPS [1 μg/ml; (Bii) ]. Flow cytometry was used to quantify surface marker expression. Data are expressed as a percentage of cells that co-express CD45 and CD11b. Representative dot plots showing the CD45 and CD11b expressing cell population plus and minus LPS treatment are also shown (Biii) . Data are pooled samples from at least five separate experiments. *** P
Figure Legend Snippet: Effects of DAMPs on surface marker expression in microglia cells . The expression of surface markers of microglia activation MHC class II (A) , and CD11c (B) were measured after 24 h exposure to the NLRP3-activating DAMPs ATP (5 mM), MSU, and CPPD [both 250 μg/ml; (Bi) ] or the PAMP LPS [1 μg/ml; (Bii) ]. Flow cytometry was used to quantify surface marker expression. Data are expressed as a percentage of cells that co-express CD45 and CD11b. Representative dot plots showing the CD45 and CD11b expressing cell population plus and minus LPS treatment are also shown (Biii) . Data are pooled samples from at least five separate experiments. *** P

Techniques Used: Marker, Expressing, Activation Assay, Flow Cytometry, Cytometry

The effects of PAMPs and DAMPs on the release of IL-1 from cultured mixed glia . Cultured mixed glia were treated (1 h) with the NLRP3-activating DAMPs ATP (5 mM), MSU, and CPPD (both 250 μg/ml) plus and minus a 24 h priming stimulus with the PAMP LPS (1 μg/ml). Release of both IL-1β (A) and IL-1α (B) were quantified by ELISA (Ai) , and processing of pro- (31 kDa) to mature (17 kDa) IL-1β in PAMP and DAMP treated cells was analyzed by Western blot (Aii) . Data are pooled samples from at least five separate experiments. ** P
Figure Legend Snippet: The effects of PAMPs and DAMPs on the release of IL-1 from cultured mixed glia . Cultured mixed glia were treated (1 h) with the NLRP3-activating DAMPs ATP (5 mM), MSU, and CPPD (both 250 μg/ml) plus and minus a 24 h priming stimulus with the PAMP LPS (1 μg/ml). Release of both IL-1β (A) and IL-1α (B) were quantified by ELISA (Ai) , and processing of pro- (31 kDa) to mature (17 kDa) IL-1β in PAMP and DAMP treated cells was analyzed by Western blot (Aii) . Data are pooled samples from at least five separate experiments. ** P

Techniques Used: Cell Culture, Enzyme-linked Immunosorbent Assay, Western Blot

Effects of DAMPs on the expression of pro-inflammatory genes in cultured mixed glia . mRNA levels of pro-inflammatory genes were measured by qPCR after 4 h exposure to the NLRP3-activating DAMPs ATP (5 mM), MSU, and CPPD (both 250 μg/ml) (Ai) or the PAMP LPS (1 μg/ml) (Aii) . Data were normalized to expression levels of the housekeeping gene SDHA across each treatment and fold change was expressed relative to basal RNA levels from untreated mixed glia. The genes analyzed were IL-1β (A) , IL-1α (B) , caspase-1 (C) , NLRP3 (D) , ASC (E) , iNOS (F) , IL-6 (G) , TNFα (H) , and CXCL1 (I) . Data are pooled samples from at least five separate experiments. ** P
Figure Legend Snippet: Effects of DAMPs on the expression of pro-inflammatory genes in cultured mixed glia . mRNA levels of pro-inflammatory genes were measured by qPCR after 4 h exposure to the NLRP3-activating DAMPs ATP (5 mM), MSU, and CPPD (both 250 μg/ml) (Ai) or the PAMP LPS (1 μg/ml) (Aii) . Data were normalized to expression levels of the housekeeping gene SDHA across each treatment and fold change was expressed relative to basal RNA levels from untreated mixed glia. The genes analyzed were IL-1β (A) , IL-1α (B) , caspase-1 (C) , NLRP3 (D) , ASC (E) , iNOS (F) , IL-6 (G) , TNFα (H) , and CXCL1 (I) . Data are pooled samples from at least five separate experiments. ** P

Techniques Used: Expressing, Cell Culture, Real-time Polymerase Chain Reaction

13) Product Images from "BOT-4-one attenuates NLRP3 inflammasome activation: NLRP3 alkylation leading to the regulation of its ATPase activity and ubiquitination"

Article Title: BOT-4-one attenuates NLRP3 inflammasome activation: NLRP3 alkylation leading to the regulation of its ATPase activity and ubiquitination

Journal: Scientific Reports

doi: 10.1038/s41598-017-15314-8

BOT-4-one inhibits NLRP3 inflammasome activation. ( a ) The molecular structure of BOT-4-one. LPS-primed BMDMs were treated with BOT-4-one or zVAD for 1 h and then stimulated with ATP, nigericin (Nig), or silica crystals (Sil). The caspase inhibitor zVAD was employed as a positive control for the general inhibition of inflammasome activations. ( b ) IL-1β (p17) and cleaved caspase-1 (Casp-1) in the supernatants (Sup) were analyzed by immunoblot. Full-length blots/gels are presented in Supplementary Fig. S9a . ( c ) LDH release in cell supernatants was measured by LDH assay. Pam3CSK4-primed BMDMs were treated with BOT-4-one or KCl for 1 h and then transfected with LPS. KCl was used as a positive control for the NLRP3 inflammasome-specific inhibition. The supernatants were analyzed by immunoblot ( d ) and LDH assay ( e ). Full-length blots/gels are presented in Supplementary Fig. S9b . ( f ) LPS-primed BMDMs were treated with BOT-4-one or zVAD for 1 h and then stimulated with ATP, nigericin (Nig), or silica crystals (Sil). IL-1β secretion in the cell supernatants was measured by ELISA. LPS-primed BMDMs were treated with BOT-4-one, MNS, or Bay11-7082 (Bay) for 1 h, and then stimulated with nigericin (Nig). The supernatants were analyzed by ELISA ( g ) and immunoblot ( h ). Full-length blots/gels are presented in Supplementary Fig. S9c . The data represent the mean ± SEM of three independent experiments; *** p
Figure Legend Snippet: BOT-4-one inhibits NLRP3 inflammasome activation. ( a ) The molecular structure of BOT-4-one. LPS-primed BMDMs were treated with BOT-4-one or zVAD for 1 h and then stimulated with ATP, nigericin (Nig), or silica crystals (Sil). The caspase inhibitor zVAD was employed as a positive control for the general inhibition of inflammasome activations. ( b ) IL-1β (p17) and cleaved caspase-1 (Casp-1) in the supernatants (Sup) were analyzed by immunoblot. Full-length blots/gels are presented in Supplementary Fig. S9a . ( c ) LDH release in cell supernatants was measured by LDH assay. Pam3CSK4-primed BMDMs were treated with BOT-4-one or KCl for 1 h and then transfected with LPS. KCl was used as a positive control for the NLRP3 inflammasome-specific inhibition. The supernatants were analyzed by immunoblot ( d ) and LDH assay ( e ). Full-length blots/gels are presented in Supplementary Fig. S9b . ( f ) LPS-primed BMDMs were treated with BOT-4-one or zVAD for 1 h and then stimulated with ATP, nigericin (Nig), or silica crystals (Sil). IL-1β secretion in the cell supernatants was measured by ELISA. LPS-primed BMDMs were treated with BOT-4-one, MNS, or Bay11-7082 (Bay) for 1 h, and then stimulated with nigericin (Nig). The supernatants were analyzed by ELISA ( g ) and immunoblot ( h ). Full-length blots/gels are presented in Supplementary Fig. S9c . The data represent the mean ± SEM of three independent experiments; *** p

Techniques Used: Activation Assay, Positive Control, Inhibition, Lactate Dehydrogenase Assay, Transfection, Enzyme-linked Immunosorbent Assay

14) Product Images from "Repositioning of the β-Blocker Carvedilol as a Novel Autophagy Inducer That Inhibits the NLRP3 Inflammasome"

Article Title: Repositioning of the β-Blocker Carvedilol as a Novel Autophagy Inducer That Inhibits the NLRP3 Inflammasome

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.01920

CVL reduced NLRP3 inflammasome activation. (A) J774A.1 macrophages were incubated for 5 h with LPS (1 μg/ml) (LPS priming) followed by incubation for 0.5 h with CVL. Cells were then incubated with CC (100 μg/ml, 24 h), MSU (100 μg/ml, 24 h), ATP (5 mM, 0.5 h), nigericin (10 μM, 0.5 h), and nano-SiO 2 (100 μg/ml, 24 h). (B) LPS-primed J774A.1 macrophages were incubated for 0.5 h with CVL followed by incubation with CC (100 μg/ml, 24 h), MSU (100 μg/ml, 24 h), and ATP (5 mM, 0.5 h). (C) LPS-primed BMDM were incubated for 0.5 h with CVL followed by incubation with CC (100 μg/ml) for an additional 24 h. (D) LPS-primed or Pam3CSK4-primed (for LPS transfection only) cells were incubated for 0.5 h with CVL followed by transfection with poly(dA/dT) (2 μg/ml, 6 h), FLA-ST (1 μg/ml, 6 h), MDP (10 μg/ml, 6 h), or LPS (2 μg/ml, 6 h). The levels of IL-1β, IL-18, NLRP3, ASC, and caspase-1 in the culture medium were measured by Western blot. The IL-1β levels in the supernatants were measured by ELISA. The Western blot results are representative of three different experiments. The ELISA data are expressed as the mean ± SD of three separate experiments. *, **, and *** indicate a significant difference at the level of p
Figure Legend Snippet: CVL reduced NLRP3 inflammasome activation. (A) J774A.1 macrophages were incubated for 5 h with LPS (1 μg/ml) (LPS priming) followed by incubation for 0.5 h with CVL. Cells were then incubated with CC (100 μg/ml, 24 h), MSU (100 μg/ml, 24 h), ATP (5 mM, 0.5 h), nigericin (10 μM, 0.5 h), and nano-SiO 2 (100 μg/ml, 24 h). (B) LPS-primed J774A.1 macrophages were incubated for 0.5 h with CVL followed by incubation with CC (100 μg/ml, 24 h), MSU (100 μg/ml, 24 h), and ATP (5 mM, 0.5 h). (C) LPS-primed BMDM were incubated for 0.5 h with CVL followed by incubation with CC (100 μg/ml) for an additional 24 h. (D) LPS-primed or Pam3CSK4-primed (for LPS transfection only) cells were incubated for 0.5 h with CVL followed by transfection with poly(dA/dT) (2 μg/ml, 6 h), FLA-ST (1 μg/ml, 6 h), MDP (10 μg/ml, 6 h), or LPS (2 μg/ml, 6 h). The levels of IL-1β, IL-18, NLRP3, ASC, and caspase-1 in the culture medium were measured by Western blot. The IL-1β levels in the supernatants were measured by ELISA. The Western blot results are representative of three different experiments. The ELISA data are expressed as the mean ± SD of three separate experiments. *, **, and *** indicate a significant difference at the level of p

Techniques Used: Activation Assay, Incubation, Transfection, Western Blot, Enzyme-linked Immunosorbent Assay

15) Product Images from "The PYRIN domain-only protein POP2 inhibits inflammasome priming and activation"

Article Title: The PYRIN domain-only protein POP2 inhibits inflammasome priming and activation

Journal: Nature Communications

doi: 10.1038/ncomms15556

POP2 inhibits inflammasome assembly and activation in mouse macrophages. ( a ) Interaction of GST-POP2 with endogenous ASC from LPS-primed THP-1 cells and iBMDM TCLs using GST as negative control and showing 10% TCL as input. ( b ) Immunoblot analysis of ASC polymerization (oligomer) in untreated or LPS/nigericin (Nig)-treated WT BMDM and POP2 BMDM after non-reversible crosslinking of pellets (P) and in TCL. ( c , d ) Flow cytometric quantification of active caspase-1 in LPS-primed WT, POP2 and Asc −/− BMDM that were treated with control (Ctrl) or ( c ) nigericin or ( d ) ATP, % FLICA + live, single cells is listed. ( e ) Immunoblot analysis of active caspase-1 p10 and IL-1β release into culture SN of control, LPS-primed and LPS-primed and MSU-treated WT, POP2 and Asc −/− BMDM. Pro-caspase-1 and pro-IL-1β expression in TCL confirms equal loading. ns indicates a cross-reactive nonspecific protein. ( f – i ) Analysis of culture SN for IL-1β release by ELISA in control (Ctrl), LPS-primed (Ctrl+), or LPS-primed and ( f ) ATP-treated, ( g ) nigericin-treated or poly(dA:dT)-transfected and ( h ) MSU, SiO 2 or CPPD or ( i ) TcdB-treated or flagellin (Fla) and Bacillus anthracis lethal toxin-transfected WT, POP2 and Asc −/− BMDM. ( h ) POP2#1 and POP2#2 BMDM represent two independent POP2 TG lines. ( j – l ) Analysis of culture SN for release of ( j ) IL-18, ( k ) LDH and ( l ) IL-1α in response to the treatment of WT, POP2 and Asc −/− BMDM as above. Between three and five independent repeats were performed for each experiment. Significance was calculated by a standard two-tailed unpaired t -test and error bars represent s.e.m., * P
Figure Legend Snippet: POP2 inhibits inflammasome assembly and activation in mouse macrophages. ( a ) Interaction of GST-POP2 with endogenous ASC from LPS-primed THP-1 cells and iBMDM TCLs using GST as negative control and showing 10% TCL as input. ( b ) Immunoblot analysis of ASC polymerization (oligomer) in untreated or LPS/nigericin (Nig)-treated WT BMDM and POP2 BMDM after non-reversible crosslinking of pellets (P) and in TCL. ( c , d ) Flow cytometric quantification of active caspase-1 in LPS-primed WT, POP2 and Asc −/− BMDM that were treated with control (Ctrl) or ( c ) nigericin or ( d ) ATP, % FLICA + live, single cells is listed. ( e ) Immunoblot analysis of active caspase-1 p10 and IL-1β release into culture SN of control, LPS-primed and LPS-primed and MSU-treated WT, POP2 and Asc −/− BMDM. Pro-caspase-1 and pro-IL-1β expression in TCL confirms equal loading. ns indicates a cross-reactive nonspecific protein. ( f – i ) Analysis of culture SN for IL-1β release by ELISA in control (Ctrl), LPS-primed (Ctrl+), or LPS-primed and ( f ) ATP-treated, ( g ) nigericin-treated or poly(dA:dT)-transfected and ( h ) MSU, SiO 2 or CPPD or ( i ) TcdB-treated or flagellin (Fla) and Bacillus anthracis lethal toxin-transfected WT, POP2 and Asc −/− BMDM. ( h ) POP2#1 and POP2#2 BMDM represent two independent POP2 TG lines. ( j – l ) Analysis of culture SN for release of ( j ) IL-18, ( k ) LDH and ( l ) IL-1α in response to the treatment of WT, POP2 and Asc −/− BMDM as above. Between three and five independent repeats were performed for each experiment. Significance was calculated by a standard two-tailed unpaired t -test and error bars represent s.e.m., * P

Techniques Used: Activation Assay, Negative Control, Flow Cytometry, Expressing, Enzyme-linked Immunosorbent Assay, Transfection, Two Tailed Test

POP2 inhibits inflammasome priming in human macrophages. ( a , b , d ) Analysis of culture SN for IL-6 release by ELISA in response to LPS, MSU crystals and nigericin in ( a ) GFP and GFP-POP2-stable THP-1 cells, ( b ) control and His-POP2-stable THP-1 cells and ( d ) control (shRNA Ctrl ) and POP2 shRNA (shRNA POP2#1 , shRNA POP2#2 ) expressing THP-1 cells. ( c , e – i ) Immunoblot analysis of phosphorylated and non-phosphorylated proteins in response to LPS treatment of ( c , f ) GFP and GFP-POP2 ( e , h , i ) control and POP2 shRNA-expressing stable THP-1 cells and ( g ) WT and POP2 BMDM for the indicated times. Tubulin protein levels were used as a loading control and GFP to detect expression of GFP and GFP-POP2. ( j ) Real-time PCR analysis of the indicated transcripts in control shRNA, POP2#1 shRNA and POP2#2 shRNA-expressing THP-1 cells in response to LPS induction presented as fold compared to untreated control shRNA cells. Three independent repeats were performed for each experiment. Significance was calculated by a standard two-tailed unpaired t -test and error bars represent s.e.m., * P
Figure Legend Snippet: POP2 inhibits inflammasome priming in human macrophages. ( a , b , d ) Analysis of culture SN for IL-6 release by ELISA in response to LPS, MSU crystals and nigericin in ( a ) GFP and GFP-POP2-stable THP-1 cells, ( b ) control and His-POP2-stable THP-1 cells and ( d ) control (shRNA Ctrl ) and POP2 shRNA (shRNA POP2#1 , shRNA POP2#2 ) expressing THP-1 cells. ( c , e – i ) Immunoblot analysis of phosphorylated and non-phosphorylated proteins in response to LPS treatment of ( c , f ) GFP and GFP-POP2 ( e , h , i ) control and POP2 shRNA-expressing stable THP-1 cells and ( g ) WT and POP2 BMDM for the indicated times. Tubulin protein levels were used as a loading control and GFP to detect expression of GFP and GFP-POP2. ( j ) Real-time PCR analysis of the indicated transcripts in control shRNA, POP2#1 shRNA and POP2#2 shRNA-expressing THP-1 cells in response to LPS induction presented as fold compared to untreated control shRNA cells. Three independent repeats were performed for each experiment. Significance was calculated by a standard two-tailed unpaired t -test and error bars represent s.e.m., * P

Techniques Used: Enzyme-linked Immunosorbent Assay, shRNA, Expressing, Real-time Polymerase Chain Reaction, Two Tailed Test

POP2 inhibits inflammasome activation in human macrophages. ( a , b ) Flow cytometric quantification of active caspase-1 in control (Ctrl) and His-POP2 stably expressing THP-1 cells in response to nigericin (Nig) treatment for the indicated times in LPS-primed cells, indicating the % FLICA + live, single cells showing ( a ) contour plots and ( b ) quantification. ( c ) Kinetic microplate assay of PI uptake by THP-1 cells stably expressing GFP or GFP-POP2 in response to LPS priming followed by nigericin treatment. ( d ) Real-time PCR analysis of POP2 transcripts in stable control (shRNA Ctrl ) and two independent POP2 shRNA (shRNA POP2#1 and shRNA POP2#2 )-expressing THP-1 cells presented as fold compared to control shRNA cells. ( e ) Flow cytometric quantification of active caspase-1 by FLICA assay in LPS-primed POP2-silenced THP-1 cells in response to nigericin (45 min). ( f – j ) Analysis of culture SN for ( f , g , i ) IL-1β and ( h , j ) IL-18 release by ELISA in LPS-primed ( f ) GFP and GFP-POP2-stable THP-1 cells, ( g , h ) control and His-POP2-stable cells and ( i , j ) control and POP2 shRNA-expressing THP-1 cells in response to the indicated activators. ( k ) Primary human macrophages were transfected with a scrambled control or two POP2-specific siRNAs were primed with LPS, and culture SNs were analysed for secreted IL-1β release in response to MSU crystals and ATP. ( l ) Immunoprecipitation (IP) of ASC or control immunoglobulin G (IgG) from LPS-primed and nigericin-treated WT, control and His-POP2-stable THP-1 cells, followed by immunoblot analysis alongside TCL. HC indicates the antibody heavy chain. Three independent repeats were performed for each experiment. Significance was calculated by a standard two-tailed unpaired t -test and error bars represent s.e.m., * P
Figure Legend Snippet: POP2 inhibits inflammasome activation in human macrophages. ( a , b ) Flow cytometric quantification of active caspase-1 in control (Ctrl) and His-POP2 stably expressing THP-1 cells in response to nigericin (Nig) treatment for the indicated times in LPS-primed cells, indicating the % FLICA + live, single cells showing ( a ) contour plots and ( b ) quantification. ( c ) Kinetic microplate assay of PI uptake by THP-1 cells stably expressing GFP or GFP-POP2 in response to LPS priming followed by nigericin treatment. ( d ) Real-time PCR analysis of POP2 transcripts in stable control (shRNA Ctrl ) and two independent POP2 shRNA (shRNA POP2#1 and shRNA POP2#2 )-expressing THP-1 cells presented as fold compared to control shRNA cells. ( e ) Flow cytometric quantification of active caspase-1 by FLICA assay in LPS-primed POP2-silenced THP-1 cells in response to nigericin (45 min). ( f – j ) Analysis of culture SN for ( f , g , i ) IL-1β and ( h , j ) IL-18 release by ELISA in LPS-primed ( f ) GFP and GFP-POP2-stable THP-1 cells, ( g , h ) control and His-POP2-stable cells and ( i , j ) control and POP2 shRNA-expressing THP-1 cells in response to the indicated activators. ( k ) Primary human macrophages were transfected with a scrambled control or two POP2-specific siRNAs were primed with LPS, and culture SNs were analysed for secreted IL-1β release in response to MSU crystals and ATP. ( l ) Immunoprecipitation (IP) of ASC or control immunoglobulin G (IgG) from LPS-primed and nigericin-treated WT, control and His-POP2-stable THP-1 cells, followed by immunoblot analysis alongside TCL. HC indicates the antibody heavy chain. Three independent repeats were performed for each experiment. Significance was calculated by a standard two-tailed unpaired t -test and error bars represent s.e.m., * P

Techniques Used: Activation Assay, Flow Cytometry, Stable Transfection, Expressing, Real-time Polymerase Chain Reaction, shRNA, Enzyme-linked Immunosorbent Assay, Transfection, Immunoprecipitation, Two Tailed Test

POP2 expression is induced in response to pro- and anti-inflammatory cytokines. ( a – f ) POP2 , POP1 and TNF transcripts as indicated were measured by real-time PCR in ( a , c , d ) primary human macrophages (MΦ). ( b , e ) THP-1 cells and ( f ) WT and POP2 BMDM treated with various pro- and anti-inflammatory PAMPs and cytokines for the indicated times. ( d , e ) Cells were pretreated with LPS or Pam 3 CSK 4 for 1 h and then further incubated in d for a total of 4 h and ( e ) 4 and 9 h in the presence of the NF-κB inhibitor Bay11-7082. ( g ) POP2 expression in BMDM isolated from individual POP2 TG mice was determined by immunoblot and densitometric quantification and correlated to the release of IL-1β in response to nigericin or the release of TNF in LPS-primed cells and was analysed by linear regression. ( h , i ) TAT-GFP or TAT-POP2 (0.28 μM) was added to the culture medium of unprimed or LPS-primed ( h ) BMDM and ( i ) iBMDM for 30 min followed by 45 min treatment with nigericin. IL-1β release was quantified by ELISA. ( j ) Proposed function of POP2 as a dual regulator that simultaneously inhibits NF-κB-mediated inflammasome priming and nucleation in macrophages in response to MSU crystals and other activators. POP2 is positioned in a negative feedback loop for inflammasome regulation since it is upregulated by IL-1β, which is blocked upon POP2 expression. For NF-κB signalling POP2 is positioned in a feed-forward regulatory loop since its inhibition of NF-κB leads to POP2 upregulation. Together, POP2 efficiently prevents cytokine release and pyroptosis. Three independent repeats were performed for each experiment in a – i . Significance was calculated by a standard two-tailed unpaired t -test, and error bars represent s.e.m., * P
Figure Legend Snippet: POP2 expression is induced in response to pro- and anti-inflammatory cytokines. ( a – f ) POP2 , POP1 and TNF transcripts as indicated were measured by real-time PCR in ( a , c , d ) primary human macrophages (MΦ). ( b , e ) THP-1 cells and ( f ) WT and POP2 BMDM treated with various pro- and anti-inflammatory PAMPs and cytokines for the indicated times. ( d , e ) Cells were pretreated with LPS or Pam 3 CSK 4 for 1 h and then further incubated in d for a total of 4 h and ( e ) 4 and 9 h in the presence of the NF-κB inhibitor Bay11-7082. ( g ) POP2 expression in BMDM isolated from individual POP2 TG mice was determined by immunoblot and densitometric quantification and correlated to the release of IL-1β in response to nigericin or the release of TNF in LPS-primed cells and was analysed by linear regression. ( h , i ) TAT-GFP or TAT-POP2 (0.28 μM) was added to the culture medium of unprimed or LPS-primed ( h ) BMDM and ( i ) iBMDM for 30 min followed by 45 min treatment with nigericin. IL-1β release was quantified by ELISA. ( j ) Proposed function of POP2 as a dual regulator that simultaneously inhibits NF-κB-mediated inflammasome priming and nucleation in macrophages in response to MSU crystals and other activators. POP2 is positioned in a negative feedback loop for inflammasome regulation since it is upregulated by IL-1β, which is blocked upon POP2 expression. For NF-κB signalling POP2 is positioned in a feed-forward regulatory loop since its inhibition of NF-κB leads to POP2 upregulation. Together, POP2 efficiently prevents cytokine release and pyroptosis. Three independent repeats were performed for each experiment in a – i . Significance was calculated by a standard two-tailed unpaired t -test, and error bars represent s.e.m., * P

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Incubation, Isolation, Mouse Assay, Enzyme-linked Immunosorbent Assay, Inhibition, Two Tailed Test

POP2 expression ameliorates inflammation in vivo . ( a ) Immunohistochemical staining of CD68 in red and POP2 in brown in inflamed human lung tissue (left and middle) showing the original magnification × 10 (left, scale bar, 50 μm) and × 40 (middle, scale bar, 10 μm) and human RA synovial lining showing the original magnification × 10 (right, scale bar, 50 μm). ( b ) Analysis of POP2 expression by flow cytometry in peripheral blood cell populations from WT and POP2 TG mice using SmartFlares ( n =2). ( c ) POP2 expression in peripheral blood-derived macrophages isolated from WT and POP2 TG mice by immunoblot. Membranes were stripped and re-probed with a β-Actin antibody as loading control ( n =5). ( d ) In vivo imaging of MPO activity of infiltrating neutrophils with a luminescent probe in response to i.p. injection with MSU crystals (3 mg; +) or PBS (−) in WT and POP2 TG mice after 4 h (left, scale bar, 1 cm), and quantification of the luminescent counts (right; n =9). ( e ) Cytokine ELISA and ( f ) immunoblot for extracellular ASC particles in cleared peritoneal exudates of WT and POP2 TG mice 8 h after MSU injection ( n =10–13). ( g ) In vivo imaging of MPO activity correlating to MSU-induced neutrophil infiltration into air pouches 7 h after MSU (3 mg per airpouch; +) or PBS (−) injection in WT and POP2 TG mice (left, scale bar, 1 cm) and signal quantification (right; n =9–10). ( h ) FACS analysis of infiltrating cells in airpouch lavage exudates of WT and POP2 TG mice 8 h after MSU injection ( n =11). ( i ) In vivo imaging of MPO activity in LPS-induced peritonitis 4 h after i.p. injection of LPS (2.5 mg kg −1 ) or PBS (left, scale bar, 1 cm) and quantification (right; n =11). ( j , k ) Cytokine ELISA and LDH quantification of ( j ) cleared peritoneal exudates and ( k ) serum of WT and POP2 TG mice 4 h after LPS injection ( n =8–19). ( l ) Survival analysis of the indicated genotypes in response to acute nigericin-induced shock following i.p. injection of nigericin (6 mg kg −1 ) in LPS-primed mice (0.4 mg kg −1 ; n =4–17) and significance was determined by asymmetrical log-rank Mantel–Cox survival test. A standard two-tailed unpaired t -test was used for all other calculations. Error bars represent s.e.m., * P
Figure Legend Snippet: POP2 expression ameliorates inflammation in vivo . ( a ) Immunohistochemical staining of CD68 in red and POP2 in brown in inflamed human lung tissue (left and middle) showing the original magnification × 10 (left, scale bar, 50 μm) and × 40 (middle, scale bar, 10 μm) and human RA synovial lining showing the original magnification × 10 (right, scale bar, 50 μm). ( b ) Analysis of POP2 expression by flow cytometry in peripheral blood cell populations from WT and POP2 TG mice using SmartFlares ( n =2). ( c ) POP2 expression in peripheral blood-derived macrophages isolated from WT and POP2 TG mice by immunoblot. Membranes were stripped and re-probed with a β-Actin antibody as loading control ( n =5). ( d ) In vivo imaging of MPO activity of infiltrating neutrophils with a luminescent probe in response to i.p. injection with MSU crystals (3 mg; +) or PBS (−) in WT and POP2 TG mice after 4 h (left, scale bar, 1 cm), and quantification of the luminescent counts (right; n =9). ( e ) Cytokine ELISA and ( f ) immunoblot for extracellular ASC particles in cleared peritoneal exudates of WT and POP2 TG mice 8 h after MSU injection ( n =10–13). ( g ) In vivo imaging of MPO activity correlating to MSU-induced neutrophil infiltration into air pouches 7 h after MSU (3 mg per airpouch; +) or PBS (−) injection in WT and POP2 TG mice (left, scale bar, 1 cm) and signal quantification (right; n =9–10). ( h ) FACS analysis of infiltrating cells in airpouch lavage exudates of WT and POP2 TG mice 8 h after MSU injection ( n =11). ( i ) In vivo imaging of MPO activity in LPS-induced peritonitis 4 h after i.p. injection of LPS (2.5 mg kg −1 ) or PBS (left, scale bar, 1 cm) and quantification (right; n =11). ( j , k ) Cytokine ELISA and LDH quantification of ( j ) cleared peritoneal exudates and ( k ) serum of WT and POP2 TG mice 4 h after LPS injection ( n =8–19). ( l ) Survival analysis of the indicated genotypes in response to acute nigericin-induced shock following i.p. injection of nigericin (6 mg kg −1 ) in LPS-primed mice (0.4 mg kg −1 ; n =4–17) and significance was determined by asymmetrical log-rank Mantel–Cox survival test. A standard two-tailed unpaired t -test was used for all other calculations. Error bars represent s.e.m., * P

Techniques Used: Expressing, In Vivo, Immunohistochemistry, Staining, Flow Cytometry, Cytometry, Mouse Assay, Derivative Assay, Isolation, In Vivo Imaging, Activity Assay, Injection, Enzyme-linked Immunosorbent Assay, FACS, Two Tailed Test

16) Product Images from "Mincle-mediated translational regulation is required for strong nitric oxide production and inflammation resolution"

Article Title: Mincle-mediated translational regulation is required for strong nitric oxide production and inflammation resolution

Journal: Nature Communications

doi: 10.1038/ncomms11322

TDM-induced nitric oxide upregulation inhibits activation of the NLRP3 inflammasome. ( a ) WT and Mincle −/− BMDMs were stimulated with LPS (L+A) or co-stimulated with LPS and TDM (L+T+A) for 12 h in the presence of the indicated chemical inhibitors, and then treated with ATP for 1 h. ELISA of released IL-1β. ( b ) Immunoblot analysis of total S -nitrosylated proteins (biotin), NLRP3 or caspase-1 in BMDMs treated with LPS or stimulated with TDM for 12 h. Below (lysate), immunoblot analysis of total lysate fractions. ( c ) Immunoblot analysis of IL-1β and caspase-1 from LPS-treated WT and iNOS −/− BMDMs, stimulated with TDM for 12 h, and then treated with ATP for 1 h or nigericin for 3 h. Data are representative of one ( a ) or two ( b , c ) independent experiments.
Figure Legend Snippet: TDM-induced nitric oxide upregulation inhibits activation of the NLRP3 inflammasome. ( a ) WT and Mincle −/− BMDMs were stimulated with LPS (L+A) or co-stimulated with LPS and TDM (L+T+A) for 12 h in the presence of the indicated chemical inhibitors, and then treated with ATP for 1 h. ELISA of released IL-1β. ( b ) Immunoblot analysis of total S -nitrosylated proteins (biotin), NLRP3 or caspase-1 in BMDMs treated with LPS or stimulated with TDM for 12 h. Below (lysate), immunoblot analysis of total lysate fractions. ( c ) Immunoblot analysis of IL-1β and caspase-1 from LPS-treated WT and iNOS −/− BMDMs, stimulated with TDM for 12 h, and then treated with ATP for 1 h or nigericin for 3 h. Data are representative of one ( a ) or two ( b , c ) independent experiments.

Techniques Used: Activation Assay, Enzyme-linked Immunosorbent Assay

TDM stimulation diminishes NLRP3-dependent inflammasome activation and IL-1β production. ( a , b ) WT and Mincle −/− BMDMs were stimulated with LPS or co-stimulated with LPS and TDM for 12 h or the indicated times and then treated with ATP for 1 h (L: LPS; T: TDM; A: ATP; UT: untreated). ( a ) Left: immunoblot analysis of proIL-1β, mature IL-1β (p17), procaspase-1 (proCasp-1) and cleaved caspase-1 (p10) in cell culture supernatants (SN) and whole-cell lysates (XT). Right: kinetic quantitative analysis of proIL-1β and mature IL-1β (p17) from the immunoblots. ( b ) Release of lactate dehydrogenase (LDH) (assessing cell death) from cells. ( c ) ELISA of released IL-1β from LPS-treated WT and Mincle −/− BMDMs, stimulated with TDM for 12 h and then treated with MSU, nigericin or poly(dA:dT) for 3 h. * P
Figure Legend Snippet: TDM stimulation diminishes NLRP3-dependent inflammasome activation and IL-1β production. ( a , b ) WT and Mincle −/− BMDMs were stimulated with LPS or co-stimulated with LPS and TDM for 12 h or the indicated times and then treated with ATP for 1 h (L: LPS; T: TDM; A: ATP; UT: untreated). ( a ) Left: immunoblot analysis of proIL-1β, mature IL-1β (p17), procaspase-1 (proCasp-1) and cleaved caspase-1 (p10) in cell culture supernatants (SN) and whole-cell lysates (XT). Right: kinetic quantitative analysis of proIL-1β and mature IL-1β (p17) from the immunoblots. ( b ) Release of lactate dehydrogenase (LDH) (assessing cell death) from cells. ( c ) ELISA of released IL-1β from LPS-treated WT and Mincle −/− BMDMs, stimulated with TDM for 12 h and then treated with MSU, nigericin or poly(dA:dT) for 3 h. * P

Techniques Used: Activation Assay, Cell Culture, Western Blot, Enzyme-linked Immunosorbent Assay

17) Product Images from "Evodiamine Augments NLRP3 Inflammasome Activation and Anti-bacterial Responses Through Inducing α-Tubulin Acetylation"

Article Title: Evodiamine Augments NLRP3 Inflammasome Activation and Anti-bacterial Responses Through Inducing α-Tubulin Acetylation

Journal: Frontiers in Pharmacology

doi: 10.3389/fphar.2019.00290

Evodiamine increased α-tubulin acetylation. BMDMs were primed with LPS (500 ng/ml) for 4 h, then treated with evodiamine (5 μM) for 1 h, followed by incubation with nigericin (5 μM) for 1 h (A,D,E) or ATP (2 mM) for 30 min (F) in the absence of LPS. (A) Representative immunofluorescence images showing α-tubulin (green) and acetylated (K40) α-tubulin (red) subcellular distributions. Nuclei (blue) were stained with Hoechst 33342. Scale bars, 2 μm. (B,C) LPS-primed BMDMs were treated with a grade does of evodiamine for 1 h (B) or evodiamine (5 μM) for indicated time periods (C) in the absence of LPS. Acetylated and total α-tubulin expression levels were determined by Western blotting. Actin was recruited as a loading control. (D) Representative images showing TOM20 (green) subcellular distribution. Scale bars, 2 μm. (E) Representative images showing acetylated α-tubulin (red) and γ-tubulin (green) subcellular distribution. The enlarged insets show the co-localization of acetylated α-tubulin and γ-tubulin in the perinuclear region. Scale bars, 10 μm. (F) Representative immunofluorescence images showing ASC (red) and γ-tubulin (green) subcellular distributions. The enlarged insets show the ASC and γ-tubulin in the perinuclear region. Scale bars, 10 μm. EVO, evodiamine.
Figure Legend Snippet: Evodiamine increased α-tubulin acetylation. BMDMs were primed with LPS (500 ng/ml) for 4 h, then treated with evodiamine (5 μM) for 1 h, followed by incubation with nigericin (5 μM) for 1 h (A,D,E) or ATP (2 mM) for 30 min (F) in the absence of LPS. (A) Representative immunofluorescence images showing α-tubulin (green) and acetylated (K40) α-tubulin (red) subcellular distributions. Nuclei (blue) were stained with Hoechst 33342. Scale bars, 2 μm. (B,C) LPS-primed BMDMs were treated with a grade does of evodiamine for 1 h (B) or evodiamine (5 μM) for indicated time periods (C) in the absence of LPS. Acetylated and total α-tubulin expression levels were determined by Western blotting. Actin was recruited as a loading control. (D) Representative images showing TOM20 (green) subcellular distribution. Scale bars, 2 μm. (E) Representative images showing acetylated α-tubulin (red) and γ-tubulin (green) subcellular distribution. The enlarged insets show the co-localization of acetylated α-tubulin and γ-tubulin in the perinuclear region. Scale bars, 10 μm. (F) Representative immunofluorescence images showing ASC (red) and γ-tubulin (green) subcellular distributions. The enlarged insets show the ASC and γ-tubulin in the perinuclear region. Scale bars, 10 μm. EVO, evodiamine.

Techniques Used: Incubation, Immunofluorescence, Staining, Expressing, Western Blot

Evodiamine promoted ATP- or nigericin-induced pyroptosis in LPS-primed macrophages. J774A.1 cells (A,D) were treated as in Figure 1B . BMDMs in (B,E,G) were treated as in Figure 1C , and other BMDMs (C,F) were treated as in Figure 1D . (A–C) Western blotting was used to assess the expression levels of indicated proteins in the cell lysates. Actin was used as a loading control for cell lysates. (D–G) Cell death was measured by staining with propidium iodide (PI) (red, staining dead cells) for 10 min. The images were captured by fluorescence microscopy, the merged images show PI fluorescence with bright-field images. One set of representative images of three independent experiments are shown (G) . Scale bars, 20 μm. PI-positive cells were quantified by counting 5 randomly chosen fields (one field per well) containing around 100 cells each. Data are shown as mean ± SD ( n = 5). One-way analysis of variance (ANOVA): P
Figure Legend Snippet: Evodiamine promoted ATP- or nigericin-induced pyroptosis in LPS-primed macrophages. J774A.1 cells (A,D) were treated as in Figure 1B . BMDMs in (B,E,G) were treated as in Figure 1C , and other BMDMs (C,F) were treated as in Figure 1D . (A–C) Western blotting was used to assess the expression levels of indicated proteins in the cell lysates. Actin was used as a loading control for cell lysates. (D–G) Cell death was measured by staining with propidium iodide (PI) (red, staining dead cells) for 10 min. The images were captured by fluorescence microscopy, the merged images show PI fluorescence with bright-field images. One set of representative images of three independent experiments are shown (G) . Scale bars, 20 μm. PI-positive cells were quantified by counting 5 randomly chosen fields (one field per well) containing around 100 cells each. Data are shown as mean ± SD ( n = 5). One-way analysis of variance (ANOVA): P

Techniques Used: Western Blot, Expressing, Staining, Fluorescence, Microscopy

Knockdown of αTAT1 attenuated evodiamine-mediated enhancement of ATP- or nigericin-induced NLRP3 inflammasome activation and pyroptosis in macrophages. (A–D) J774A.1 cells and BMDMs were knocked down by small-interfering RNA (siRNA) targeting αTAT1 gene (αTAT1 siRNA). Negative control siRNA (NC siRNA) was recruited as a knockdown control. 72 h after knockdown, J774A.1 cells (A) and BMDMs (B) were lysed and analyzed by Western blotting. Actin was used as a loading control for cell lysates. After αTAT1 knockdown, J774A.1 cells (C) and BMDMs (D) were primed with LPS (500 ng/ml) for 4 h, then treated with evodiamine (5 μM) for 1 h. Representative immunofluorescence images showing acetylated α-tubulin (red) subcellular distribution. Nuclei (blue) were revealed by Hoechst 33342. The images for acetylated α-tubulin and nuclei were captured, respectively, and merged together. Scale bars, 10 μm. (E–H) J774A.1 cells (E,G) and BMDMs (F,H) were treated with αTAT1 siRNA as in a. Cells were primed with LPS (500 ng/ml) for 4 h and then pre-treated with evodiamine (5 μM) for 1 h, followed by incubation with ATP (3 mM) for 1 h (E) or nigericin (5 μM) for 1 h (F) in the absence of LPS. (E,F) Cell death was measured by staining with propidium iodide (PI) (red, staining dead cells) and Hoechst 33342 (blue, staining total cells) together for 10 min. PI-positive cells were quantified by counting 5 randomly chosen fields (one field per well) containing around 100 cells each. (G,H) The levels of soluble IL-1β in the culture supernatants were detected by cytometric bead array assay. The experiments were performed three times independently. Data are shown as mean ± SD ( n = 5). ∗ P
Figure Legend Snippet: Knockdown of αTAT1 attenuated evodiamine-mediated enhancement of ATP- or nigericin-induced NLRP3 inflammasome activation and pyroptosis in macrophages. (A–D) J774A.1 cells and BMDMs were knocked down by small-interfering RNA (siRNA) targeting αTAT1 gene (αTAT1 siRNA). Negative control siRNA (NC siRNA) was recruited as a knockdown control. 72 h after knockdown, J774A.1 cells (A) and BMDMs (B) were lysed and analyzed by Western blotting. Actin was used as a loading control for cell lysates. After αTAT1 knockdown, J774A.1 cells (C) and BMDMs (D) were primed with LPS (500 ng/ml) for 4 h, then treated with evodiamine (5 μM) for 1 h. Representative immunofluorescence images showing acetylated α-tubulin (red) subcellular distribution. Nuclei (blue) were revealed by Hoechst 33342. The images for acetylated α-tubulin and nuclei were captured, respectively, and merged together. Scale bars, 10 μm. (E–H) J774A.1 cells (E,G) and BMDMs (F,H) were treated with αTAT1 siRNA as in a. Cells were primed with LPS (500 ng/ml) for 4 h and then pre-treated with evodiamine (5 μM) for 1 h, followed by incubation with ATP (3 mM) for 1 h (E) or nigericin (5 μM) for 1 h (F) in the absence of LPS. (E,F) Cell death was measured by staining with propidium iodide (PI) (red, staining dead cells) and Hoechst 33342 (blue, staining total cells) together for 10 min. PI-positive cells were quantified by counting 5 randomly chosen fields (one field per well) containing around 100 cells each. (G,H) The levels of soluble IL-1β in the culture supernatants were detected by cytometric bead array assay. The experiments were performed three times independently. Data are shown as mean ± SD ( n = 5). ∗ P

Techniques Used: Activation Assay, Small Interfering RNA, Negative Control, Western Blot, Immunofluorescence, Incubation, Staining

Evodiamine-mediated enhancement of ATP- or nigericin-induced NLRP3 inflammasome activation was attenuated by blocking acetylation of α-tubulin. (A) LPS-primed BMDMs were pre-treated with resveratrol (5 μM), NAD + (10 μM) or ciliobrevin A (30 μM) for 30 min, and then incubated with evodiamine (5 μM) for 1 h, or followed by stimulation with nigericin (5 μM) for 1 h. Representative immunofluorescence images showing acetylated α-tubulin (red) subcellular distribution. Nuclei (blue) were revealed by Hoechst 33342. The images for acetylated α-tubulin and nuclei were captured, respectively, and merged together. Scale bars, 10 μm. (B) LPS-primed BMDMs were pre-treated with resveratrol (5 μM) for 30 min, and then incubated with indicated dose of evodiamine for 1 h, followed by stimulation with ATP (2 mM) for 30 min. Representative images showing ASC (green) subcellular distribution. The images for ASC and nuclei were captured, respectively, and merged together. Yellow arrows indicate ASC specks and the enlarged inset showing cells with an ASC speck. Scale bars, 20 μm. (C) Percentages of cells with an ASC speck relative to the total number of cells from 5 random fields (one field per well) each containing ∼200 cells. Data are shown as mean ± SD ( n = 5). ∗ P
Figure Legend Snippet: Evodiamine-mediated enhancement of ATP- or nigericin-induced NLRP3 inflammasome activation was attenuated by blocking acetylation of α-tubulin. (A) LPS-primed BMDMs were pre-treated with resveratrol (5 μM), NAD + (10 μM) or ciliobrevin A (30 μM) for 30 min, and then incubated with evodiamine (5 μM) for 1 h, or followed by stimulation with nigericin (5 μM) for 1 h. Representative immunofluorescence images showing acetylated α-tubulin (red) subcellular distribution. Nuclei (blue) were revealed by Hoechst 33342. The images for acetylated α-tubulin and nuclei were captured, respectively, and merged together. Scale bars, 10 μm. (B) LPS-primed BMDMs were pre-treated with resveratrol (5 μM) for 30 min, and then incubated with indicated dose of evodiamine for 1 h, followed by stimulation with ATP (2 mM) for 30 min. Representative images showing ASC (green) subcellular distribution. The images for ASC and nuclei were captured, respectively, and merged together. Yellow arrows indicate ASC specks and the enlarged inset showing cells with an ASC speck. Scale bars, 20 μm. (C) Percentages of cells with an ASC speck relative to the total number of cells from 5 random fields (one field per well) each containing ∼200 cells. Data are shown as mean ± SD ( n = 5). ∗ P

Techniques Used: Activation Assay, Blocking Assay, Incubation, Immunofluorescence

Evodiamine specifically promoted NLRP3 inflammasome activation in macrophages. (A) The chemical strcuture of evodiamine. (B–D) J774A.1 cells (B) and bone marrow-derived macrophages (BMDMs) (C,D) were treated as described in Section “ Materials and Methods .” Western blotting was used to assess the expression levels of indicated proteins in the cell lysates and culture supernatants, respectively. Actin was used as a loading control for cell lysates. (E) Human THP-1 macrophages were induced by PMA (500 nM) treatment for 16 h. The cells were then primed with LPS (1 μg/ml) for 4 h, and pre-treated with series concentrations of evodiamine for 1 h, followed by incubation with nigericin (20 μM) for 1 h in the absence of LPS. The levels of soluble IL-1β in the culture supernatants were detected by cytometric bead array (CBA) assay. Data are shown as mean ± SD ( n = 5). One-way analysis of variance (ANOVA): P
Figure Legend Snippet: Evodiamine specifically promoted NLRP3 inflammasome activation in macrophages. (A) The chemical strcuture of evodiamine. (B–D) J774A.1 cells (B) and bone marrow-derived macrophages (BMDMs) (C,D) were treated as described in Section “ Materials and Methods .” Western blotting was used to assess the expression levels of indicated proteins in the cell lysates and culture supernatants, respectively. Actin was used as a loading control for cell lysates. (E) Human THP-1 macrophages were induced by PMA (500 nM) treatment for 16 h. The cells were then primed with LPS (1 μg/ml) for 4 h, and pre-treated with series concentrations of evodiamine for 1 h, followed by incubation with nigericin (20 μM) for 1 h in the absence of LPS. The levels of soluble IL-1β in the culture supernatants were detected by cytometric bead array (CBA) assay. Data are shown as mean ± SD ( n = 5). One-way analysis of variance (ANOVA): P

Techniques Used: Activation Assay, Derivative Assay, Western Blot, Expressing, Incubation, Crocin Bleaching Assay

18) Product Images from "Artemisia Extract Suppresses NLRP3 and AIM2 Inflammasome Activation by Inhibition of ASC Phosphorylation"

Article Title: Artemisia Extract Suppresses NLRP3 and AIM2 Inflammasome Activation by Inhibition of ASC Phosphorylation

Journal: Mediators of Inflammation

doi: 10.1155/2018/6054069

Inhibitory effect of APO on ASC oligomerization. LPS-primed BMDMs were pretreated with APO or KCl (150 mM), and ASC oligomerization in the cells was determined by Western blotting upon stimulation with (a) 5 mM ATP for 1 h, (b) 150 μ g/mL silica for 3 h, (c) 10 μ M nigericin for 1 h, (d) 2 μ g/mL poly (dA:dT) for 1 h, and (e) 1.5 μ g/mL flagellin for 3 h. β -Actin is used as an internal control. (f) LPS-primed BMDMs were stimulated with 5 mM ATP for 1 h, 150 μ g/mL silica for 3 h, 10 μ M nigericin for 1 h, 2 μ g/mL poly (dA:dT) for 1 h, and 1.5 μ g/mL flagellin for 3 h and stained with anti-ASC antibody for ASC specks (red) and by DAPI for nuclei (blue). ASC specks were marked with arrows, and (g) cells with ASC specks are presented as a percentage of the positive cells. Data were expressed as the mean ± SEM ( n = 3). Statistical analysis was performed using Student's t -test. ∗ p
Figure Legend Snippet: Inhibitory effect of APO on ASC oligomerization. LPS-primed BMDMs were pretreated with APO or KCl (150 mM), and ASC oligomerization in the cells was determined by Western blotting upon stimulation with (a) 5 mM ATP for 1 h, (b) 150 μ g/mL silica for 3 h, (c) 10 μ M nigericin for 1 h, (d) 2 μ g/mL poly (dA:dT) for 1 h, and (e) 1.5 μ g/mL flagellin for 3 h. β -Actin is used as an internal control. (f) LPS-primed BMDMs were stimulated with 5 mM ATP for 1 h, 150 μ g/mL silica for 3 h, 10 μ M nigericin for 1 h, 2 μ g/mL poly (dA:dT) for 1 h, and 1.5 μ g/mL flagellin for 3 h and stained with anti-ASC antibody for ASC specks (red) and by DAPI for nuclei (blue). ASC specks were marked with arrows, and (g) cells with ASC specks are presented as a percentage of the positive cells. Data were expressed as the mean ± SEM ( n = 3). Statistical analysis was performed using Student's t -test. ∗ p

Techniques Used: Western Blot, Staining

Effect of APO on inflammasome components. LPS-primed BMDMs (a–d, f) and nonprimed BMDMs (e, g) were pretreated with APO or zVAD (20 μ M) and stimulated with (a) 150 μ g/mL silica for 3 h, (b) 10 μ M nigericin for 1 h, (c) 5 mM ATP for 1 h, (d-e) 2 μ g/mL poly (dA:dT) for 1 h, and (f-g) 1.5 μ g/mL flagellin for 3 h. Mature IL-1 β and caspase-1 cleavage were measured in supernatant (Sup). NLRP3, pro-IL-1 β , procaspase-1, and ASC were measured in whole cell extract (WCE) by immunoblotting. β -Actin was used as an internal control. LPS, lipopolysaccharide; BMDMs, bone marrow-derived macrophages; APO, A. princeps extract; IL, interleukin; Nig, nigericin.
Figure Legend Snippet: Effect of APO on inflammasome components. LPS-primed BMDMs (a–d, f) and nonprimed BMDMs (e, g) were pretreated with APO or zVAD (20 μ M) and stimulated with (a) 150 μ g/mL silica for 3 h, (b) 10 μ M nigericin for 1 h, (c) 5 mM ATP for 1 h, (d-e) 2 μ g/mL poly (dA:dT) for 1 h, and (f-g) 1.5 μ g/mL flagellin for 3 h. Mature IL-1 β and caspase-1 cleavage were measured in supernatant (Sup). NLRP3, pro-IL-1 β , procaspase-1, and ASC were measured in whole cell extract (WCE) by immunoblotting. β -Actin was used as an internal control. LPS, lipopolysaccharide; BMDMs, bone marrow-derived macrophages; APO, A. princeps extract; IL, interleukin; Nig, nigericin.

Techniques Used: Derivative Assay

Effect of APO on cell viability and IL-1 β secretion in LPS-primed BMDMs. (a) BMDMs were treated with the indicated concentration of APO for 24 h. Cell viability was measured by the LDH assay. (b–f) LPS-primed BMDMs were pretreated with APO or zVAD (20 μ M), and IL-1 β secretion was determined by ELISA upon stimulation with (b) 150 μ g/mL silica for 3 h, (c) 10 μ M nigericin for 1 h, (d) 5 mM ATP for 1 h, (e) 2 μ g/mL poly (dA:dT) for 1 h, and (f) 1.5 μ g/mL flagellin for 3 h. (g) TNF- α release from LPS-primed BMDMs was determined upon stimulation with 10 μ M nigericin for 1 h. (h) The impact of APO on MSU-induced IL-1 β production in mice. Data were expressed as the mean ± SEM ( n = 3). Statistical analysis was performed using Student's t -test. ∗ p
Figure Legend Snippet: Effect of APO on cell viability and IL-1 β secretion in LPS-primed BMDMs. (a) BMDMs were treated with the indicated concentration of APO for 24 h. Cell viability was measured by the LDH assay. (b–f) LPS-primed BMDMs were pretreated with APO or zVAD (20 μ M), and IL-1 β secretion was determined by ELISA upon stimulation with (b) 150 μ g/mL silica for 3 h, (c) 10 μ M nigericin for 1 h, (d) 5 mM ATP for 1 h, (e) 2 μ g/mL poly (dA:dT) for 1 h, and (f) 1.5 μ g/mL flagellin for 3 h. (g) TNF- α release from LPS-primed BMDMs was determined upon stimulation with 10 μ M nigericin for 1 h. (h) The impact of APO on MSU-induced IL-1 β production in mice. Data were expressed as the mean ± SEM ( n = 3). Statistical analysis was performed using Student's t -test. ∗ p

Techniques Used: Concentration Assay, Lactate Dehydrogenase Assay, Enzyme-linked Immunosorbent Assay, Mouse Assay

19) Product Images from "Divergent effects of Tlr9 deletion in experimental late venous thrombosis resolution and vein wall injury"

Article Title: Divergent effects of Tlr9 deletion in experimental late venous thrombosis resolution and vein wall injury

Journal: Thrombosis and haemostasis

doi: 10.1160/TH14-12-1031

BMMØ cell culture gene expression results at 8 hours after exposure. A) WT BMMØ was stimulated with either ODN 1826 (CpG), MSU 300 μg/mL, or control media (ctl); or RNA 1 μg/mL. necrotic cells (50%). Data shown are from
Figure Legend Snippet: BMMØ cell culture gene expression results at 8 hours after exposure. A) WT BMMØ was stimulated with either ODN 1826 (CpG), MSU 300 μg/mL, or control media (ctl); or RNA 1 μg/mL. necrotic cells (50%). Data shown are from

Techniques Used: Cell Culture, Expressing, CTL Assay

20) Product Images from "Targeting ASC in NLRP3 inflammasome by caffeic acid phenethyl ester: a novel strategy to treat acute gout"

Article Title: Targeting ASC in NLRP3 inflammasome by caffeic acid phenethyl ester: a novel strategy to treat acute gout

Journal: Scientific Reports

doi: 10.1038/srep38622

CAPE blocks the interaction between NLRP3 and ASC. ( A ) The chemical structure of CAPE and the proposed molecular docking model for CAPE binding to ASC. ( B ) Electrostatic surface binding model for CAPE and ASC. Red: negative charge, blue: positive charge. ( C,D ) BMDMs were primed with LPS ( C , 500 ng/ml; D , 100 ng/ml) for 4 hr. Then, the cells were treated with CAPE for 1 hr, followed by stimulation with MSU (500 μg/ml) for 5 hr or ATP (5 mM) for 1 hr. Cell lysates were immunoprepitated with anti-ASC antibody followed by immunoblotting as indicated. Representative data from at least two independent experiments are presented.
Figure Legend Snippet: CAPE blocks the interaction between NLRP3 and ASC. ( A ) The chemical structure of CAPE and the proposed molecular docking model for CAPE binding to ASC. ( B ) Electrostatic surface binding model for CAPE and ASC. Red: negative charge, blue: positive charge. ( C,D ) BMDMs were primed with LPS ( C , 500 ng/ml; D , 100 ng/ml) for 4 hr. Then, the cells were treated with CAPE for 1 hr, followed by stimulation with MSU (500 μg/ml) for 5 hr or ATP (5 mM) for 1 hr. Cell lysates were immunoprepitated with anti-ASC antibody followed by immunoblotting as indicated. Representative data from at least two independent experiments are presented.

Techniques Used: Binding Assay

21) Product Images from "Repositioning of the β-Blocker Carvedilol as a Novel Autophagy Inducer That Inhibits the NLRP3 Inflammasome"

Article Title: Repositioning of the β-Blocker Carvedilol as a Novel Autophagy Inducer That Inhibits the NLRP3 Inflammasome

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.01920

CVL reduced NLRP3 inflammasome activation. (A) J774A.1 macrophages were incubated for 5 h with LPS (1 μg/ml) (LPS priming) followed by incubation for 0.5 h with CVL. Cells were then incubated with CC (100 μg/ml, 24 h), MSU (100 μg/ml, 24 h), ATP (5 mM, 0.5 h), nigericin (10 μM, 0.5 h), and nano-SiO 2 (100 μg/ml, 24 h). (B) LPS-primed J774A.1 macrophages were incubated for 0.5 h with CVL followed by incubation with CC (100 μg/ml, 24 h), MSU (100 μg/ml, 24 h), and ATP (5 mM, 0.5 h). (C) LPS-primed BMDM were incubated for 0.5 h with CVL followed by incubation with CC (100 μg/ml) for an additional 24 h. (D) LPS-primed or Pam3CSK4-primed (for LPS transfection only) cells were incubated for 0.5 h with CVL followed by transfection with poly(dA/dT) (2 μg/ml, 6 h), FLA-ST (1 μg/ml, 6 h), MDP (10 μg/ml, 6 h), or LPS (2 μg/ml, 6 h). The levels of IL-1β, IL-18, NLRP3, ASC, and caspase-1 in the culture medium were measured by Western blot. The IL-1β levels in the supernatants were measured by ELISA. The Western blot results are representative of three different experiments. The ELISA data are expressed as the mean ± SD of three separate experiments. *, **, and *** indicate a significant difference at the level of p
Figure Legend Snippet: CVL reduced NLRP3 inflammasome activation. (A) J774A.1 macrophages were incubated for 5 h with LPS (1 μg/ml) (LPS priming) followed by incubation for 0.5 h with CVL. Cells were then incubated with CC (100 μg/ml, 24 h), MSU (100 μg/ml, 24 h), ATP (5 mM, 0.5 h), nigericin (10 μM, 0.5 h), and nano-SiO 2 (100 μg/ml, 24 h). (B) LPS-primed J774A.1 macrophages were incubated for 0.5 h with CVL followed by incubation with CC (100 μg/ml, 24 h), MSU (100 μg/ml, 24 h), and ATP (5 mM, 0.5 h). (C) LPS-primed BMDM were incubated for 0.5 h with CVL followed by incubation with CC (100 μg/ml) for an additional 24 h. (D) LPS-primed or Pam3CSK4-primed (for LPS transfection only) cells were incubated for 0.5 h with CVL followed by transfection with poly(dA/dT) (2 μg/ml, 6 h), FLA-ST (1 μg/ml, 6 h), MDP (10 μg/ml, 6 h), or LPS (2 μg/ml, 6 h). The levels of IL-1β, IL-18, NLRP3, ASC, and caspase-1 in the culture medium were measured by Western blot. The IL-1β levels in the supernatants were measured by ELISA. The Western blot results are representative of three different experiments. The ELISA data are expressed as the mean ± SD of three separate experiments. *, **, and *** indicate a significant difference at the level of p

Techniques Used: Activation Assay, Incubation, Transfection, Western Blot, Enzyme-linked Immunosorbent Assay

22) Product Images from "Uric acid crystals induces placental inflammation and alters trophoblast function via an IL-1-dependent pathway: implication for FGR"

Article Title: Uric acid crystals induces placental inflammation and alters trophoblast function via an IL-1-dependent pathway: implication for FGR

Journal: Journal of immunology (Baltimore, Md. : 1950)

doi: 10.4049/jimmunol.1601179

Histological analysis of IL-1β staining in the placenta following MSU crystals treatment.
Figure Legend Snippet: Histological analysis of IL-1β staining in the placenta following MSU crystals treatment.

Techniques Used: Staining

23) Product Images from "Artemisia Extract Suppresses NLRP3 and AIM2 Inflammasome Activation by Inhibition of ASC Phosphorylation"

Article Title: Artemisia Extract Suppresses NLRP3 and AIM2 Inflammasome Activation by Inhibition of ASC Phosphorylation

Journal: Mediators of Inflammation

doi: 10.1155/2018/6054069

Effect of APO on cell viability and IL-1 β secretion in LPS-primed BMDMs. (a) BMDMs were treated with the indicated concentration of APO for 24 h. Cell viability was measured by the LDH assay. (b–f) LPS-primed BMDMs were pretreated with APO or zVAD (20 μ M), and IL-1 β secretion was determined by ELISA upon stimulation with (b) 150 μ g/mL silica for 3 h, (c) 10 μ M nigericin for 1 h, (d) 5 mM ATP for 1 h, (e) 2 μ g/mL poly (dA:dT) for 1 h, and (f) 1.5 μ g/mL flagellin for 3 h. (g) TNF- α release from LPS-primed BMDMs was determined upon stimulation with 10 μ M nigericin for 1 h. (h) The impact of APO on MSU-induced IL-1 β production in mice. Data were expressed as the mean ± SEM ( n = 3). Statistical analysis was performed using Student's t -test. ∗ p
Figure Legend Snippet: Effect of APO on cell viability and IL-1 β secretion in LPS-primed BMDMs. (a) BMDMs were treated with the indicated concentration of APO for 24 h. Cell viability was measured by the LDH assay. (b–f) LPS-primed BMDMs were pretreated with APO or zVAD (20 μ M), and IL-1 β secretion was determined by ELISA upon stimulation with (b) 150 μ g/mL silica for 3 h, (c) 10 μ M nigericin for 1 h, (d) 5 mM ATP for 1 h, (e) 2 μ g/mL poly (dA:dT) for 1 h, and (f) 1.5 μ g/mL flagellin for 3 h. (g) TNF- α release from LPS-primed BMDMs was determined upon stimulation with 10 μ M nigericin for 1 h. (h) The impact of APO on MSU-induced IL-1 β production in mice. Data were expressed as the mean ± SEM ( n = 3). Statistical analysis was performed using Student's t -test. ∗ p

Techniques Used: Concentration Assay, Lactate Dehydrogenase Assay, Enzyme-linked Immunosorbent Assay, Mouse Assay

24) Product Images from "Genipin inhibits NLRP3 and NLRC4 inflammasome activation via autophagy suppression"

Article Title: Genipin inhibits NLRP3 and NLRC4 inflammasome activation via autophagy suppression

Journal: Scientific Reports

doi: 10.1038/srep17935

Genipin inhibits autophagy-dependent inflammasome activation. ( A ) Analysis of the cell death phenotype by Annexin-V/PI staining. LPS-primed BMDMs were pretreated with genipin and then stimulated with nigericin or Salmonella . The cells were stained with Annexin-V and PI. The percentage of cells positive for both Annexin V and PI is shown. ( B ) LPS-primed BMDMs were incubated with genipin and/or 5 μM wortm annin for 1 h and then stimulated with nigericin or Salmonella . The cells were fixed, permeabilized and stained for LC3. LC3 is shown in green, and cell nuclei are shown in blue (DAPI). ( C , D ) LPS-primed BMDMs were incubated with genipin and/or wortmannin and then stimulated with nigericin or Salmonella . The cell lysates were immunoblotted for LC3, β-tubulin or GAPDH. ( E ) LPS-primed BMDMs were incubated with genipin and/or wortmannin and then stimulated with nigericin. The cross-linked pellets were immunoblotted for ASC. ( F,G ) LPS-primed BMDMs were incubated with genipin and/or wortmannin and then stimulated with nigericin or flagellin. IL-1β secretion was measured by ELISA. ( H ) BMDMs were transfected with either siRNA targeting ATG5 or scrambled siRNA (NEG). Forty-eight hours after transfection, the cells were primed with LPS for 4 h and then stimulated with genipin before nigericin or Salmonella challenge. IL-1β secretion was measured by ELISA. ( I ) LPS-primed BMDMs were incubated with the indicated doses of torkinib or rapamycin for 1 h and then stimulated with nigericin. IL-1β secretion was measured by ELISA. The data are from three independent experiments conducted in triplicate. * P
Figure Legend Snippet: Genipin inhibits autophagy-dependent inflammasome activation. ( A ) Analysis of the cell death phenotype by Annexin-V/PI staining. LPS-primed BMDMs were pretreated with genipin and then stimulated with nigericin or Salmonella . The cells were stained with Annexin-V and PI. The percentage of cells positive for both Annexin V and PI is shown. ( B ) LPS-primed BMDMs were incubated with genipin and/or 5 μM wortm annin for 1 h and then stimulated with nigericin or Salmonella . The cells were fixed, permeabilized and stained for LC3. LC3 is shown in green, and cell nuclei are shown in blue (DAPI). ( C , D ) LPS-primed BMDMs were incubated with genipin and/or wortmannin and then stimulated with nigericin or Salmonella . The cell lysates were immunoblotted for LC3, β-tubulin or GAPDH. ( E ) LPS-primed BMDMs were incubated with genipin and/or wortmannin and then stimulated with nigericin. The cross-linked pellets were immunoblotted for ASC. ( F,G ) LPS-primed BMDMs were incubated with genipin and/or wortmannin and then stimulated with nigericin or flagellin. IL-1β secretion was measured by ELISA. ( H ) BMDMs were transfected with either siRNA targeting ATG5 or scrambled siRNA (NEG). Forty-eight hours after transfection, the cells were primed with LPS for 4 h and then stimulated with genipin before nigericin or Salmonella challenge. IL-1β secretion was measured by ELISA. ( I ) LPS-primed BMDMs were incubated with the indicated doses of torkinib or rapamycin for 1 h and then stimulated with nigericin. IL-1β secretion was measured by ELISA. The data are from three independent experiments conducted in triplicate. * P

Techniques Used: Activation Assay, Staining, Incubation, Enzyme-linked Immunosorbent Assay, Transfection

Genipin prevents NLRP3-mediated, but not NLRC4-mediated, ASC oligomerization. ( A ) LPS-primed BMDMs were pretreated with the indicated doses of genipin for 1 h and then stimulated with nigericin. The cells were extracted with 0.5% Triton X-100, and Triton-insoluble pellets were cross-linked with DSS. The cross-linked pellets were immunoblotted for ASC as described in the Materials and Methods section. The culture supernatants were immunoblotted for caspase-1. ( B ) LPS-primed wild-type (WT) or NLRP3 KO BMDMs were pretreated with the indicated doses of genipin, followed by Listeria or nigericin challenge. The cell pellets cross-linked with DSS were immunoblotted for ASC, and cell supernatants were immunoblotted for caspase-1. ( C ) LPS-primed BMDMs were stimulated with nigericin, Listeria or Salmonella in the absence or presence of genipin. The cells were fixed, permeabilized and stained for ASC (green). DAPI was used to label nuclei (blue). ( D ) The percentage of cells containing ASC speckles was quantified from three different view fields. ( E ) LPS-primed BMDMs were either not treated or pretreated with genipin, followed by Salmonella challenge. The cross-linked pellets were immunoblotted for ASC, and the culture supernatants were immunoblotted for caspase-1. The data are representative of three independent experiments. ** P
Figure Legend Snippet: Genipin prevents NLRP3-mediated, but not NLRC4-mediated, ASC oligomerization. ( A ) LPS-primed BMDMs were pretreated with the indicated doses of genipin for 1 h and then stimulated with nigericin. The cells were extracted with 0.5% Triton X-100, and Triton-insoluble pellets were cross-linked with DSS. The cross-linked pellets were immunoblotted for ASC as described in the Materials and Methods section. The culture supernatants were immunoblotted for caspase-1. ( B ) LPS-primed wild-type (WT) or NLRP3 KO BMDMs were pretreated with the indicated doses of genipin, followed by Listeria or nigericin challenge. The cell pellets cross-linked with DSS were immunoblotted for ASC, and cell supernatants were immunoblotted for caspase-1. ( C ) LPS-primed BMDMs were stimulated with nigericin, Listeria or Salmonella in the absence or presence of genipin. The cells were fixed, permeabilized and stained for ASC (green). DAPI was used to label nuclei (blue). ( D ) The percentage of cells containing ASC speckles was quantified from three different view fields. ( E ) LPS-primed BMDMs were either not treated or pretreated with genipin, followed by Salmonella challenge. The cross-linked pellets were immunoblotted for ASC, and the culture supernatants were immunoblotted for caspase-1. The data are representative of three independent experiments. ** P

Techniques Used: Staining

Genipin inhibits UCP2 expression and induces ROS accumulation. ( A ) LPS-primed BMDMs were pretreated with genipin and then stimulated with ATP or Salmonella . The cells were fixed, permeabilized and stained for UCP2 (green). DAPI was used to label nuclei (blue). ( B ) LPS-primed BMDMs stimulated with genipin and/or Salmonella were lysed and blotted for UCP2. ( C ) LPS-primed BMDMs were stimulated with genipin and/or ATP and/or Salmonella . The cells were incubated with MitoSox Red for 10 min and analyzed using a FACSAria flow cytometer. ( D ) MitoSox MFI was analyzed using FCS express. ( E ) LPS-primed BMDMs were pretreated with genipin and/or NAC at the indicated doses and then stimulated with nigericin. IL-1β release in the cell supernatants was determined by ELISA. The data are representative of three independent experiments. ** P
Figure Legend Snippet: Genipin inhibits UCP2 expression and induces ROS accumulation. ( A ) LPS-primed BMDMs were pretreated with genipin and then stimulated with ATP or Salmonella . The cells were fixed, permeabilized and stained for UCP2 (green). DAPI was used to label nuclei (blue). ( B ) LPS-primed BMDMs stimulated with genipin and/or Salmonella were lysed and blotted for UCP2. ( C ) LPS-primed BMDMs were stimulated with genipin and/or ATP and/or Salmonella . The cells were incubated with MitoSox Red for 10 min and analyzed using a FACSAria flow cytometer. ( D ) MitoSox MFI was analyzed using FCS express. ( E ) LPS-primed BMDMs were pretreated with genipin and/or NAC at the indicated doses and then stimulated with nigericin. IL-1β release in the cell supernatants was determined by ELISA. The data are representative of three independent experiments. ** P

Techniques Used: Expressing, Staining, Incubation, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay

Genipin inhibits NLRP3 and NLRC4 inflammasome-mediated IL-1β secretion and caspase-1 activation in mouse macrophages. LPS-primed BMDMs were incubated with genipin (200 μM unless otherwise indicated) or DMSO for 1 h, followed by treatment with various NLRP3 or NLRC4 inflammasome agonists. Culture supernatants were analyzed for IL-1β and TNF-α by ELISA. Precipitated cell supernatants (Sup) or cell extracts (Lysate) were immunoblotted using various antibodies. ( A ) IL-1β secretion in BMDMs stimulated with the indicated doses of genipin and ATP (5 mM, 1 h). ( B ) IL-1β secretion in BMDMs stimulated with genipin plus ATP, nigericin (Nige, 20 μM, 1 h), MSU (100 μg/ml, 4 h) or Listeria (MOI = 20, 4 h). ( C ) IL-1β secretion in BMDMs stimulated with the indicated doses of genipin and Salmonella (MOI = 20, 4 h). ( D ) IL-1β secretion in BMDMs transfected with 20 μg/ml flagellin or BSA for 8 h using Lipofectamine 2000. ( E ) TNF-α secretion in BMDMs stimulated with genipin plus ATP, nigericin, MSU, Listeria , Salmonella or flagellin. ( F ) Cell supernatants and cell extracts immunoblotted for caspase-1, IL-1β and ASC. GAPDH and β-tubulin served as loading controls. The data are representative of three independent experiments. ** P
Figure Legend Snippet: Genipin inhibits NLRP3 and NLRC4 inflammasome-mediated IL-1β secretion and caspase-1 activation in mouse macrophages. LPS-primed BMDMs were incubated with genipin (200 μM unless otherwise indicated) or DMSO for 1 h, followed by treatment with various NLRP3 or NLRC4 inflammasome agonists. Culture supernatants were analyzed for IL-1β and TNF-α by ELISA. Precipitated cell supernatants (Sup) or cell extracts (Lysate) were immunoblotted using various antibodies. ( A ) IL-1β secretion in BMDMs stimulated with the indicated doses of genipin and ATP (5 mM, 1 h). ( B ) IL-1β secretion in BMDMs stimulated with genipin plus ATP, nigericin (Nige, 20 μM, 1 h), MSU (100 μg/ml, 4 h) or Listeria (MOI = 20, 4 h). ( C ) IL-1β secretion in BMDMs stimulated with the indicated doses of genipin and Salmonella (MOI = 20, 4 h). ( D ) IL-1β secretion in BMDMs transfected with 20 μg/ml flagellin or BSA for 8 h using Lipofectamine 2000. ( E ) TNF-α secretion in BMDMs stimulated with genipin plus ATP, nigericin, MSU, Listeria , Salmonella or flagellin. ( F ) Cell supernatants and cell extracts immunoblotted for caspase-1, IL-1β and ASC. GAPDH and β-tubulin served as loading controls. The data are representative of three independent experiments. ** P

Techniques Used: Activation Assay, Incubation, Enzyme-linked Immunosorbent Assay, Transfection

25) Product Images from "NLRP3-Inflammasome Activating DAMPs Stimulate an Inflammatory Response in Glia in the Absence of Priming Which Contributes to Brain Inflammation after Injury"

Article Title: NLRP3-Inflammasome Activating DAMPs Stimulate an Inflammatory Response in Glia in the Absence of Priming Which Contributes to Brain Inflammation after Injury

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2012.00288

DAMPs induce the release of proteases from cultured mixed glia . Cultured mixed glia were treated with the NLRP3-activating DAMPs ATP (5 mM), MSU, and CPPD (both 250 μg/ml) or the PAMP LPS (1 μg/ml) for 24 h. Supernatants were analyzed for the release of mature Cathepsin B (28–30 kDa) by Western blot (A) . To measure the release of martix metalloproteases by zymography all samples were loaded neat onto gels except for one of the ATP-treated supernatants (+*) which was diluted 1:3 (B) . Blots and gels shown are representative of at least five separate experiments.
Figure Legend Snippet: DAMPs induce the release of proteases from cultured mixed glia . Cultured mixed glia were treated with the NLRP3-activating DAMPs ATP (5 mM), MSU, and CPPD (both 250 μg/ml) or the PAMP LPS (1 μg/ml) for 24 h. Supernatants were analyzed for the release of mature Cathepsin B (28–30 kDa) by Western blot (A) . To measure the release of martix metalloproteases by zymography all samples were loaded neat onto gels except for one of the ATP-treated supernatants (+*) which was diluted 1:3 (B) . Blots and gels shown are representative of at least five separate experiments.

Techniques Used: Cell Culture, Western Blot, Zymography

Effects of DAMPs on pro-inflammatory protein levels in cultured mixed glia . Protein levels of pro-inflammatory mediators were measured by specific ELISAs after 24 h (A–D) or 4 h (E) exposure to the NLRP3-activating DAMPs ATP (5 mM), MSU, and CPPD [both 250 μg/ml; (Ai) ] or the PAMP LPS [1 μg/ml; (Aii) ]. The proteins analyzed were IL-1β (A) , IL-1α (B) , IL-6 (C,E) , CXCL1 (D,E) . Data are pooled samples from at least five separate experiments. *** P
Figure Legend Snippet: Effects of DAMPs on pro-inflammatory protein levels in cultured mixed glia . Protein levels of pro-inflammatory mediators were measured by specific ELISAs after 24 h (A–D) or 4 h (E) exposure to the NLRP3-activating DAMPs ATP (5 mM), MSU, and CPPD [both 250 μg/ml; (Ai) ] or the PAMP LPS [1 μg/ml; (Aii) ]. The proteins analyzed were IL-1β (A) , IL-1α (B) , IL-6 (C,E) , CXCL1 (D,E) . Data are pooled samples from at least five separate experiments. *** P

Techniques Used: Cell Culture

Effects of DAMPs on surface marker expression in microglia cells . The expression of surface markers of microglia activation MHC class II (A) , and CD11c (B) were measured after 24 h exposure to the NLRP3-activating DAMPs ATP (5 mM), MSU, and CPPD [both 250 μg/ml; (Bi) ] or the PAMP LPS [1 μg/ml; (Bii) ]. Flow cytometry was used to quantify surface marker expression. Data are expressed as a percentage of cells that co-express CD45 and CD11b. Representative dot plots showing the CD45 and CD11b expressing cell population plus and minus LPS treatment are also shown (Biii) . Data are pooled samples from at least five separate experiments. *** P
Figure Legend Snippet: Effects of DAMPs on surface marker expression in microglia cells . The expression of surface markers of microglia activation MHC class II (A) , and CD11c (B) were measured after 24 h exposure to the NLRP3-activating DAMPs ATP (5 mM), MSU, and CPPD [both 250 μg/ml; (Bi) ] or the PAMP LPS [1 μg/ml; (Bii) ]. Flow cytometry was used to quantify surface marker expression. Data are expressed as a percentage of cells that co-express CD45 and CD11b. Representative dot plots showing the CD45 and CD11b expressing cell population plus and minus LPS treatment are also shown (Biii) . Data are pooled samples from at least five separate experiments. *** P

Techniques Used: Marker, Expressing, Activation Assay, Flow Cytometry, Cytometry

The effects of PAMPs and DAMPs on the release of IL-1 from cultured mixed glia . Cultured mixed glia were treated (1 h) with the NLRP3-activating DAMPs ATP (5 mM), MSU, and CPPD (both 250 μg/ml) plus and minus a 24 h priming stimulus with the PAMP LPS (1 μg/ml). Release of both IL-1β (A) and IL-1α (B) were quantified by ELISA (Ai) , and processing of pro- (31 kDa) to mature (17 kDa) IL-1β in PAMP and DAMP treated cells was analyzed by Western blot (Aii) . Data are pooled samples from at least five separate experiments. ** P
Figure Legend Snippet: The effects of PAMPs and DAMPs on the release of IL-1 from cultured mixed glia . Cultured mixed glia were treated (1 h) with the NLRP3-activating DAMPs ATP (5 mM), MSU, and CPPD (both 250 μg/ml) plus and minus a 24 h priming stimulus with the PAMP LPS (1 μg/ml). Release of both IL-1β (A) and IL-1α (B) were quantified by ELISA (Ai) , and processing of pro- (31 kDa) to mature (17 kDa) IL-1β in PAMP and DAMP treated cells was analyzed by Western blot (Aii) . Data are pooled samples from at least five separate experiments. ** P

Techniques Used: Cell Culture, Enzyme-linked Immunosorbent Assay, Western Blot

Effects of DAMPs on the expression of pro-inflammatory genes in cultured mixed glia . mRNA levels of pro-inflammatory genes were measured by qPCR after 4 h exposure to the NLRP3-activating DAMPs ATP (5 mM), MSU, and CPPD (both 250 μg/ml) (Ai) or the PAMP LPS (1 μg/ml) (Aii) . Data were normalized to expression levels of the housekeeping gene SDHA across each treatment and fold change was expressed relative to basal RNA levels from untreated mixed glia. The genes analyzed were IL-1β (A) , IL-1α (B) , caspase-1 (C) , NLRP3 (D) , ASC (E) , iNOS (F) , IL-6 (G) , TNFα (H) , and CXCL1 (I) . Data are pooled samples from at least five separate experiments. ** P
Figure Legend Snippet: Effects of DAMPs on the expression of pro-inflammatory genes in cultured mixed glia . mRNA levels of pro-inflammatory genes were measured by qPCR after 4 h exposure to the NLRP3-activating DAMPs ATP (5 mM), MSU, and CPPD (both 250 μg/ml) (Ai) or the PAMP LPS (1 μg/ml) (Aii) . Data were normalized to expression levels of the housekeeping gene SDHA across each treatment and fold change was expressed relative to basal RNA levels from untreated mixed glia. The genes analyzed were IL-1β (A) , IL-1α (B) , caspase-1 (C) , NLRP3 (D) , ASC (E) , iNOS (F) , IL-6 (G) , TNFα (H) , and CXCL1 (I) . Data are pooled samples from at least five separate experiments. ** P

Techniques Used: Expressing, Cell Culture, Real-time Polymerase Chain Reaction

26) Product Images from "Repositioning of the β-Blocker Carvedilol as a Novel Autophagy Inducer That Inhibits the NLRP3 Inflammasome"

Article Title: Repositioning of the β-Blocker Carvedilol as a Novel Autophagy Inducer That Inhibits the NLRP3 Inflammasome

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.01920

CVL reduced NLRP3 inflammasome activation. (A) J774A.1 macrophages were incubated for 5 h with LPS (1 μg/ml) (LPS priming) followed by incubation for 0.5 h with CVL. Cells were then incubated with CC (100 μg/ml, 24 h), MSU (100 μg/ml, 24 h), ATP (5 mM, 0.5 h), nigericin (10 μM, 0.5 h), and nano-SiO 2 (100 μg/ml, 24 h). (B) LPS-primed J774A.1 macrophages were incubated for 0.5 h with CVL followed by incubation with CC (100 μg/ml, 24 h), MSU (100 μg/ml, 24 h), and ATP (5 mM, 0.5 h). (C) LPS-primed BMDM were incubated for 0.5 h with CVL followed by incubation with CC (100 μg/ml) for an additional 24 h. (D) LPS-primed or Pam3CSK4-primed (for LPS transfection only) cells were incubated for 0.5 h with CVL followed by transfection with poly(dA/dT) (2 μg/ml, 6 h), FLA-ST (1 μg/ml, 6 h), MDP (10 μg/ml, 6 h), or LPS (2 μg/ml, 6 h). The levels of IL-1β, IL-18, NLRP3, ASC, and caspase-1 in the culture medium were measured by Western blot. The IL-1β levels in the supernatants were measured by ELISA. The Western blot results are representative of three different experiments. The ELISA data are expressed as the mean ± SD of three separate experiments. *, **, and *** indicate a significant difference at the level of p
Figure Legend Snippet: CVL reduced NLRP3 inflammasome activation. (A) J774A.1 macrophages were incubated for 5 h with LPS (1 μg/ml) (LPS priming) followed by incubation for 0.5 h with CVL. Cells were then incubated with CC (100 μg/ml, 24 h), MSU (100 μg/ml, 24 h), ATP (5 mM, 0.5 h), nigericin (10 μM, 0.5 h), and nano-SiO 2 (100 μg/ml, 24 h). (B) LPS-primed J774A.1 macrophages were incubated for 0.5 h with CVL followed by incubation with CC (100 μg/ml, 24 h), MSU (100 μg/ml, 24 h), and ATP (5 mM, 0.5 h). (C) LPS-primed BMDM were incubated for 0.5 h with CVL followed by incubation with CC (100 μg/ml) for an additional 24 h. (D) LPS-primed or Pam3CSK4-primed (for LPS transfection only) cells were incubated for 0.5 h with CVL followed by transfection with poly(dA/dT) (2 μg/ml, 6 h), FLA-ST (1 μg/ml, 6 h), MDP (10 μg/ml, 6 h), or LPS (2 μg/ml, 6 h). The levels of IL-1β, IL-18, NLRP3, ASC, and caspase-1 in the culture medium were measured by Western blot. The IL-1β levels in the supernatants were measured by ELISA. The Western blot results are representative of three different experiments. The ELISA data are expressed as the mean ± SD of three separate experiments. *, **, and *** indicate a significant difference at the level of p

Techniques Used: Activation Assay, Incubation, Transfection, Western Blot, Enzyme-linked Immunosorbent Assay

27) Product Images from "Repositioning of the β-Blocker Carvedilol as a Novel Autophagy Inducer That Inhibits the NLRP3 Inflammasome"

Article Title: Repositioning of the β-Blocker Carvedilol as a Novel Autophagy Inducer That Inhibits the NLRP3 Inflammasome

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.01920

CVL reduced NLRP3 inflammasome activation. (A) J774A.1 macrophages were incubated for 5 h with LPS (1 μg/ml) (LPS priming) followed by incubation for 0.5 h with CVL. Cells were then incubated with CC (100 μg/ml, 24 h), MSU (100 μg/ml, 24 h), ATP (5 mM, 0.5 h), nigericin (10 μM, 0.5 h), and nano-SiO 2 (100 μg/ml, 24 h). (B) LPS-primed J774A.1 macrophages were incubated for 0.5 h with CVL followed by incubation with CC (100 μg/ml, 24 h), MSU (100 μg/ml, 24 h), and ATP (5 mM, 0.5 h). (C) LPS-primed BMDM were incubated for 0.5 h with CVL followed by incubation with CC (100 μg/ml) for an additional 24 h. (D) LPS-primed or Pam3CSK4-primed (for LPS transfection only) cells were incubated for 0.5 h with CVL followed by transfection with poly(dA/dT) (2 μg/ml, 6 h), FLA-ST (1 μg/ml, 6 h), MDP (10 μg/ml, 6 h), or LPS (2 μg/ml, 6 h). The levels of IL-1β, IL-18, NLRP3, ASC, and caspase-1 in the culture medium were measured by Western blot. The IL-1β levels in the supernatants were measured by ELISA. The Western blot results are representative of three different experiments. The ELISA data are expressed as the mean ± SD of three separate experiments. *, **, and *** indicate a significant difference at the level of p
Figure Legend Snippet: CVL reduced NLRP3 inflammasome activation. (A) J774A.1 macrophages were incubated for 5 h with LPS (1 μg/ml) (LPS priming) followed by incubation for 0.5 h with CVL. Cells were then incubated with CC (100 μg/ml, 24 h), MSU (100 μg/ml, 24 h), ATP (5 mM, 0.5 h), nigericin (10 μM, 0.5 h), and nano-SiO 2 (100 μg/ml, 24 h). (B) LPS-primed J774A.1 macrophages were incubated for 0.5 h with CVL followed by incubation with CC (100 μg/ml, 24 h), MSU (100 μg/ml, 24 h), and ATP (5 mM, 0.5 h). (C) LPS-primed BMDM were incubated for 0.5 h with CVL followed by incubation with CC (100 μg/ml) for an additional 24 h. (D) LPS-primed or Pam3CSK4-primed (for LPS transfection only) cells were incubated for 0.5 h with CVL followed by transfection with poly(dA/dT) (2 μg/ml, 6 h), FLA-ST (1 μg/ml, 6 h), MDP (10 μg/ml, 6 h), or LPS (2 μg/ml, 6 h). The levels of IL-1β, IL-18, NLRP3, ASC, and caspase-1 in the culture medium were measured by Western blot. The IL-1β levels in the supernatants were measured by ELISA. The Western blot results are representative of three different experiments. The ELISA data are expressed as the mean ± SD of three separate experiments. *, **, and *** indicate a significant difference at the level of p

Techniques Used: Activation Assay, Incubation, Transfection, Western Blot, Enzyme-linked Immunosorbent Assay

28) Product Images from "Recombinant human proteoglycan-4 reduces phagocytosis of urate crystals and downstream nuclear factor kappa B and inflammasome activation and production of cytokines and chemokines in human and murine macrophages"

Article Title: Recombinant human proteoglycan-4 reduces phagocytosis of urate crystals and downstream nuclear factor kappa B and inflammasome activation and production of cytokines and chemokines in human and murine macrophages

Journal: Arthritis Research & Therapy

doi: 10.1186/s13075-018-1693-x

Time and treatment-dependent phagocytosis of monosodium urate monohydrate (MSU) crystals by differentiated human THP-1 macrophages using flow cytometry and impact of recombinant human proteoglycan-4 (rhPRG4) or bovine submaxillary mucin (BSM) treatments following 2 and 4-h incubations. Quantitative determination of MSU phagocytosis was performed using the percentage of cells in the P2 region of interest. Data represent the mean ± S.D. of four independent experiments. * p
Figure Legend Snippet: Time and treatment-dependent phagocytosis of monosodium urate monohydrate (MSU) crystals by differentiated human THP-1 macrophages using flow cytometry and impact of recombinant human proteoglycan-4 (rhPRG4) or bovine submaxillary mucin (BSM) treatments following 2 and 4-h incubations. Quantitative determination of MSU phagocytosis was performed using the percentage of cells in the P2 region of interest. Data represent the mean ± S.D. of four independent experiments. * p

Techniques Used: Flow Cytometry, Cytometry, Recombinant

Impact of recombinant human proteoglycan-4 (rhPRG4) treatment on monosodium urate monohydrate (MSU) crystal-induced expression and production of proinflammatory cytokines and chemokines and nuclear factor kappa b (NFκB) p65 subunit nuclear translocation in THP-1 macrophages. Cytokines included interleukin-1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α). Chemokines included interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1). Gene expression data are presented as fold induction of proinflammatory cytokines and chemokines gene expression compared to control untreated THP-1 macrophages. THP-1 macrophages were treated with MSU crystals (100μg/ml) ± rhPRG4 (100μg/ml) or bovine submaxillary mucin (BSM; 25μg/ml) for 6 h ( a through d ). NFκB p65 subunit nuclear translocation in THP-1 macrophages was performed at 1 h following MSU challenge (100μg/ml). Gene expression studies were performed at 6 h ( f through i ) and cytokine and chemokine media concentrations were determined at 24 h ( j through m ). Data represent the mean ± S.D. of three to four independent experiments with duplicate wells per group. *p
Figure Legend Snippet: Impact of recombinant human proteoglycan-4 (rhPRG4) treatment on monosodium urate monohydrate (MSU) crystal-induced expression and production of proinflammatory cytokines and chemokines and nuclear factor kappa b (NFκB) p65 subunit nuclear translocation in THP-1 macrophages. Cytokines included interleukin-1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α). Chemokines included interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1). Gene expression data are presented as fold induction of proinflammatory cytokines and chemokines gene expression compared to control untreated THP-1 macrophages. THP-1 macrophages were treated with MSU crystals (100μg/ml) ± rhPRG4 (100μg/ml) or bovine submaxillary mucin (BSM; 25μg/ml) for 6 h ( a through d ). NFκB p65 subunit nuclear translocation in THP-1 macrophages was performed at 1 h following MSU challenge (100μg/ml). Gene expression studies were performed at 6 h ( f through i ) and cytokine and chemokine media concentrations were determined at 24 h ( j through m ). Data represent the mean ± S.D. of three to four independent experiments with duplicate wells per group. *p

Techniques Used: Recombinant, Expressing, Translocation Assay

Impact of recombinant human proteoglycan-4 (rhPRG4) treatment on monosodium urate monohydrate (MSU) crystal-induced NLRP3 inflammasome activation in THP-1 macrophages. THP-1 macrophages were treated with 100μg/ml MSU in the absence or presence of rhPRG4 (100 and 200μg/ml) for 12 h. H 2 O 2 (5 mM) was used as a positive control. Data represent the mean ± S.D. of three independent experiments. * p
Figure Legend Snippet: Impact of recombinant human proteoglycan-4 (rhPRG4) treatment on monosodium urate monohydrate (MSU) crystal-induced NLRP3 inflammasome activation in THP-1 macrophages. THP-1 macrophages were treated with 100μg/ml MSU in the absence or presence of rhPRG4 (100 and 200μg/ml) for 12 h. H 2 O 2 (5 mM) was used as a positive control. Data represent the mean ± S.D. of three independent experiments. * p

Techniques Used: Recombinant, Activation Assay, Positive Control

29) Product Images from "Uric acid crystals induces placental inflammation and alters trophoblast function via an IL-1-dependent pathway: implication for FGR"

Article Title: Uric acid crystals induces placental inflammation and alters trophoblast function via an IL-1-dependent pathway: implication for FGR

Journal: Journal of immunology (Baltimore, Md. : 1950)

doi: 10.4049/jimmunol.1601179

Histological analysis of IL-1β staining in the placenta following MSU crystals treatment.
Figure Legend Snippet: Histological analysis of IL-1β staining in the placenta following MSU crystals treatment.

Techniques Used: Staining

30) Product Images from "Uric acid crystals induces placental inflammation and alters trophoblast function via an IL-1-dependent pathway: implication for FGR"

Article Title: Uric acid crystals induces placental inflammation and alters trophoblast function via an IL-1-dependent pathway: implication for FGR

Journal: Journal of immunology (Baltimore, Md. : 1950)

doi: 10.4049/jimmunol.1601179

Histological analysis of CD68+ macrophage in the placenta following MSU crystals treatment.
Figure Legend Snippet: Histological analysis of CD68+ macrophage in the placenta following MSU crystals treatment.

Techniques Used:

31) Product Images from "The PYRIN domain-only protein POP2 inhibits inflammasome priming and activation"

Article Title: The PYRIN domain-only protein POP2 inhibits inflammasome priming and activation

Journal: Nature Communications

doi: 10.1038/ncomms15556

POP2 inhibits inflammasome assembly and activation in mouse macrophages. ( a ) Interaction of GST-POP2 with endogenous ASC from LPS-primed THP-1 cells and iBMDM TCLs using GST as negative control and showing 10% TCL as input. ( b ) Immunoblot analysis of ASC polymerization (oligomer) in untreated or LPS/nigericin (Nig)-treated WT BMDM and POP2 BMDM after non-reversible crosslinking of pellets (P) and in TCL. ( c , d ) Flow cytometric quantification of active caspase-1 in LPS-primed WT, POP2 and Asc −/− BMDM that were treated with control (Ctrl) or ( c ) nigericin or ( d ) ATP, % FLICA + live, single cells is listed. ( e ) Immunoblot analysis of active caspase-1 p10 and IL-1β release into culture SN of control, LPS-primed and LPS-primed and MSU-treated WT, POP2 and Asc −/− BMDM. Pro-caspase-1 and pro-IL-1β expression in TCL confirms equal loading. ns indicates a cross-reactive nonspecific protein. ( f – i ) Analysis of culture SN for IL-1β release by ELISA in control (Ctrl), LPS-primed (Ctrl+), or LPS-primed and ( f ) ATP-treated, ( g ) nigericin-treated or poly(dA:dT)-transfected and ( h ) MSU, SiO 2 or CPPD or ( i ) TcdB-treated or flagellin (Fla) and Bacillus anthracis lethal toxin-transfected WT, POP2 and Asc −/− BMDM. ( h ) POP2#1 and POP2#2 BMDM represent two independent POP2 TG lines. ( j – l ) Analysis of culture SN for release of ( j ) IL-18, ( k ) LDH and ( l ) IL-1α in response to the treatment of WT, POP2 and Asc −/− BMDM as above. Between three and five independent repeats were performed for each experiment. Significance was calculated by a standard two-tailed unpaired t -test and error bars represent s.e.m., * P
Figure Legend Snippet: POP2 inhibits inflammasome assembly and activation in mouse macrophages. ( a ) Interaction of GST-POP2 with endogenous ASC from LPS-primed THP-1 cells and iBMDM TCLs using GST as negative control and showing 10% TCL as input. ( b ) Immunoblot analysis of ASC polymerization (oligomer) in untreated or LPS/nigericin (Nig)-treated WT BMDM and POP2 BMDM after non-reversible crosslinking of pellets (P) and in TCL. ( c , d ) Flow cytometric quantification of active caspase-1 in LPS-primed WT, POP2 and Asc −/− BMDM that were treated with control (Ctrl) or ( c ) nigericin or ( d ) ATP, % FLICA + live, single cells is listed. ( e ) Immunoblot analysis of active caspase-1 p10 and IL-1β release into culture SN of control, LPS-primed and LPS-primed and MSU-treated WT, POP2 and Asc −/− BMDM. Pro-caspase-1 and pro-IL-1β expression in TCL confirms equal loading. ns indicates a cross-reactive nonspecific protein. ( f – i ) Analysis of culture SN for IL-1β release by ELISA in control (Ctrl), LPS-primed (Ctrl+), or LPS-primed and ( f ) ATP-treated, ( g ) nigericin-treated or poly(dA:dT)-transfected and ( h ) MSU, SiO 2 or CPPD or ( i ) TcdB-treated or flagellin (Fla) and Bacillus anthracis lethal toxin-transfected WT, POP2 and Asc −/− BMDM. ( h ) POP2#1 and POP2#2 BMDM represent two independent POP2 TG lines. ( j – l ) Analysis of culture SN for release of ( j ) IL-18, ( k ) LDH and ( l ) IL-1α in response to the treatment of WT, POP2 and Asc −/− BMDM as above. Between three and five independent repeats were performed for each experiment. Significance was calculated by a standard two-tailed unpaired t -test and error bars represent s.e.m., * P

Techniques Used: Activation Assay, Negative Control, Flow Cytometry, Expressing, Enzyme-linked Immunosorbent Assay, Transfection, Two Tailed Test

32) Product Images from "The PYRIN domain-only protein POP2 inhibits inflammasome priming and activation"

Article Title: The PYRIN domain-only protein POP2 inhibits inflammasome priming and activation

Journal: Nature Communications

doi: 10.1038/ncomms15556

POP2 inhibits inflammasome assembly and activation in mouse macrophages. ( a ) Interaction of GST-POP2 with endogenous ASC from LPS-primed THP-1 cells and iBMDM TCLs using GST as negative control and showing 10% TCL as input. ( b ) Immunoblot analysis of ASC polymerization (oligomer) in untreated or LPS/nigericin (Nig)-treated WT BMDM and POP2 BMDM after non-reversible crosslinking of pellets (P) and in TCL. ( c , d ) Flow cytometric quantification of active caspase-1 in LPS-primed WT, POP2 and Asc −/− BMDM that were treated with control (Ctrl) or ( c ) nigericin or ( d ) ATP, % FLICA + live, single cells is listed. ( e ) Immunoblot analysis of active caspase-1 p10 and IL-1β release into culture SN of control, LPS-primed and LPS-primed and MSU-treated WT, POP2 and Asc −/− BMDM. Pro-caspase-1 and pro-IL-1β expression in TCL confirms equal loading. ns indicates a cross-reactive nonspecific protein. ( f – i ) Analysis of culture SN for IL-1β release by ELISA in control (Ctrl), LPS-primed (Ctrl+), or LPS-primed and ( f ) ATP-treated, ( g ) nigericin-treated or poly(dA:dT)-transfected and ( h ) MSU, SiO 2 or CPPD or ( i ) TcdB-treated or flagellin (Fla) and Bacillus anthracis lethal toxin-transfected WT, POP2 and Asc −/− BMDM. ( h ) POP2#1 and POP2#2 BMDM represent two independent POP2 TG lines. ( j – l ) Analysis of culture SN for release of ( j ) IL-18, ( k ) LDH and ( l ) IL-1α in response to the treatment of WT, POP2 and Asc −/− BMDM as above. Between three and five independent repeats were performed for each experiment. Significance was calculated by a standard two-tailed unpaired t -test and error bars represent s.e.m., * P
Figure Legend Snippet: POP2 inhibits inflammasome assembly and activation in mouse macrophages. ( a ) Interaction of GST-POP2 with endogenous ASC from LPS-primed THP-1 cells and iBMDM TCLs using GST as negative control and showing 10% TCL as input. ( b ) Immunoblot analysis of ASC polymerization (oligomer) in untreated or LPS/nigericin (Nig)-treated WT BMDM and POP2 BMDM after non-reversible crosslinking of pellets (P) and in TCL. ( c , d ) Flow cytometric quantification of active caspase-1 in LPS-primed WT, POP2 and Asc −/− BMDM that were treated with control (Ctrl) or ( c ) nigericin or ( d ) ATP, % FLICA + live, single cells is listed. ( e ) Immunoblot analysis of active caspase-1 p10 and IL-1β release into culture SN of control, LPS-primed and LPS-primed and MSU-treated WT, POP2 and Asc −/− BMDM. Pro-caspase-1 and pro-IL-1β expression in TCL confirms equal loading. ns indicates a cross-reactive nonspecific protein. ( f – i ) Analysis of culture SN for IL-1β release by ELISA in control (Ctrl), LPS-primed (Ctrl+), or LPS-primed and ( f ) ATP-treated, ( g ) nigericin-treated or poly(dA:dT)-transfected and ( h ) MSU, SiO 2 or CPPD or ( i ) TcdB-treated or flagellin (Fla) and Bacillus anthracis lethal toxin-transfected WT, POP2 and Asc −/− BMDM. ( h ) POP2#1 and POP2#2 BMDM represent two independent POP2 TG lines. ( j – l ) Analysis of culture SN for release of ( j ) IL-18, ( k ) LDH and ( l ) IL-1α in response to the treatment of WT, POP2 and Asc −/− BMDM as above. Between three and five independent repeats were performed for each experiment. Significance was calculated by a standard two-tailed unpaired t -test and error bars represent s.e.m., * P

Techniques Used: Activation Assay, Negative Control, Flow Cytometry, Expressing, Enzyme-linked Immunosorbent Assay, Transfection, Two Tailed Test

33) Product Images from "Artemisia Extract Suppresses NLRP3 and AIM2 Inflammasome Activation by Inhibition of ASC Phosphorylation"

Article Title: Artemisia Extract Suppresses NLRP3 and AIM2 Inflammasome Activation by Inhibition of ASC Phosphorylation

Journal: Mediators of Inflammation

doi: 10.1155/2018/6054069

Effect of APO on cell viability and IL-1 β secretion in LPS-primed BMDMs. (a) BMDMs were treated with the indicated concentration of APO for 24 h. Cell viability was measured by the LDH assay. (b–f) LPS-primed BMDMs were pretreated with APO or zVAD (20 μ M), and IL-1 β secretion was determined by ELISA upon stimulation with (b) 150 μ g/mL silica for 3 h, (c) 10 μ M nigericin for 1 h, (d) 5 mM ATP for 1 h, (e) 2 μ g/mL poly (dA:dT) for 1 h, and (f) 1.5 μ g/mL flagellin for 3 h. (g) TNF- α release from LPS-primed BMDMs was determined upon stimulation with 10 μ M nigericin for 1 h. (h) The impact of APO on MSU-induced IL-1 β production in mice. Data were expressed as the mean ± SEM ( n = 3). Statistical analysis was performed using Student's t -test. ∗ p
Figure Legend Snippet: Effect of APO on cell viability and IL-1 β secretion in LPS-primed BMDMs. (a) BMDMs were treated with the indicated concentration of APO for 24 h. Cell viability was measured by the LDH assay. (b–f) LPS-primed BMDMs were pretreated with APO or zVAD (20 μ M), and IL-1 β secretion was determined by ELISA upon stimulation with (b) 150 μ g/mL silica for 3 h, (c) 10 μ M nigericin for 1 h, (d) 5 mM ATP for 1 h, (e) 2 μ g/mL poly (dA:dT) for 1 h, and (f) 1.5 μ g/mL flagellin for 3 h. (g) TNF- α release from LPS-primed BMDMs was determined upon stimulation with 10 μ M nigericin for 1 h. (h) The impact of APO on MSU-induced IL-1 β production in mice. Data were expressed as the mean ± SEM ( n = 3). Statistical analysis was performed using Student's t -test. ∗ p

Techniques Used: Concentration Assay, Lactate Dehydrogenase Assay, Enzyme-linked Immunosorbent Assay, Mouse Assay

34) Product Images from "Uric acid crystals induces placental inflammation and alters trophoblast function via an IL-1-dependent pathway: implication for FGR"

Article Title: Uric acid crystals induces placental inflammation and alters trophoblast function via an IL-1-dependent pathway: implication for FGR

Journal: Journal of immunology (Baltimore, Md. : 1950)

doi: 10.4049/jimmunol.1601179

Histological analysis of IL-1β staining in the placenta following MSU crystals treatment.
Figure Legend Snippet: Histological analysis of IL-1β staining in the placenta following MSU crystals treatment.

Techniques Used: Staining

35) Product Images from "Repositioning of the β-Blocker Carvedilol as a Novel Autophagy Inducer That Inhibits the NLRP3 Inflammasome"

Article Title: Repositioning of the β-Blocker Carvedilol as a Novel Autophagy Inducer That Inhibits the NLRP3 Inflammasome

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.01920

CVL inhibited the NLRP3 inflammasome by enhancing autophagy. (A) J774A.1 macrophages were incubated for 0–24 h with CVL (20 μM). The levels of LC3, p62, and ATG5 in the cell lysates were measured by Western blot. (B) J774A.1 macrophages were incubated with CVL (20 μM, 12 h) or rapamycin (100 nM, 4 h). Accumulation of the fluorescent signals of MDC and AO was measured by confocal microscopy. (C) LPS-primed J774A.1 macrophages were incubated for 0.5 h with 3-MA (5 mM) and CVL (20 μM) followed by incubation with CC (100 μg/ml) for an additional 24 h. The levels of IL-1β in the culture medium were measured by ELISA. (D) LPS-primed wild-type (scramble) and LC3-knockdown J774A.1 macrophages (clones #1 and #2) were incubated for 0.5 h with CVL (20 μM) followed by incubation with CC (100 μg/ml) or MSU (100 μg/ml) for an additional 24 h. The levels of IL-1β in the culture medium were measured by ELISA. The expression levels of LC3 in the wild-type and LC3-knockdown J774A.1 macrophages were measured by Western blot. (E,F) LPS-primed wild-type and LC3-knockdown J774A.1 macrophages were incubated for 0.5 h with CVL (20 μM) followed by incubation with MSU (100 μg/ml) for an additional 24 h. Mitochondrial integrity was measured by staining with MitoTracker Deep Red and MitoTracker Green (E) , and mitochondrial ROS was measured by staining with MitoSOX (F) . The MFI of Mitotracker Deep Red are expressed as the means ± SD for three separate experiments. The Western blot results are representative of three different experiments. The ELISA data are expressed as the mean ± SD of three separate experiments. * and *** indicate a significant difference at the level of p
Figure Legend Snippet: CVL inhibited the NLRP3 inflammasome by enhancing autophagy. (A) J774A.1 macrophages were incubated for 0–24 h with CVL (20 μM). The levels of LC3, p62, and ATG5 in the cell lysates were measured by Western blot. (B) J774A.1 macrophages were incubated with CVL (20 μM, 12 h) or rapamycin (100 nM, 4 h). Accumulation of the fluorescent signals of MDC and AO was measured by confocal microscopy. (C) LPS-primed J774A.1 macrophages were incubated for 0.5 h with 3-MA (5 mM) and CVL (20 μM) followed by incubation with CC (100 μg/ml) for an additional 24 h. The levels of IL-1β in the culture medium were measured by ELISA. (D) LPS-primed wild-type (scramble) and LC3-knockdown J774A.1 macrophages (clones #1 and #2) were incubated for 0.5 h with CVL (20 μM) followed by incubation with CC (100 μg/ml) or MSU (100 μg/ml) for an additional 24 h. The levels of IL-1β in the culture medium were measured by ELISA. The expression levels of LC3 in the wild-type and LC3-knockdown J774A.1 macrophages were measured by Western blot. (E,F) LPS-primed wild-type and LC3-knockdown J774A.1 macrophages were incubated for 0.5 h with CVL (20 μM) followed by incubation with MSU (100 μg/ml) for an additional 24 h. Mitochondrial integrity was measured by staining with MitoTracker Deep Red and MitoTracker Green (E) , and mitochondrial ROS was measured by staining with MitoSOX (F) . The MFI of Mitotracker Deep Red are expressed as the means ± SD for three separate experiments. The Western blot results are representative of three different experiments. The ELISA data are expressed as the mean ± SD of three separate experiments. * and *** indicate a significant difference at the level of p

Techniques Used: Incubation, Western Blot, Confocal Microscopy, Enzyme-linked Immunosorbent Assay, Expressing, Staining

36) Product Images from "Lentinan from shiitake selectively attenuates AIM2 and non-canonical inflammasome activation while inducing pro-inflammatory cytokine production"

Article Title: Lentinan from shiitake selectively attenuates AIM2 and non-canonical inflammasome activation while inducing pro-inflammatory cytokine production

Journal: Scientific Reports

doi: 10.1038/s41598-017-01462-4

Lentinan on NLRC4 or AIM2 inflammasome activation. LPS-primed BMDMs were treated with the indicated dosage of lentinan (LNT) with/without flagellin ( A ) or dsDNA ( B ). Secretion of caspase-1 (Casp1) was analyzed by immunoblotting, and IL-1β or IL-18 secretion was measured by ELISA. ( C ) Secretion of IL-1β secretion was confirmed by immunoblotting. ( D ) For cytotoxicity, BMDMs were treated with the indicated dosages of LNT, and cell number was measured by an automated cell counter. Triton x-100 (1%, Triton) treatment led to cell death. All immunoblot data shown are representative of at least three independent experiments. Bar graph presents the mean ± SD.
Figure Legend Snippet: Lentinan on NLRC4 or AIM2 inflammasome activation. LPS-primed BMDMs were treated with the indicated dosage of lentinan (LNT) with/without flagellin ( A ) or dsDNA ( B ). Secretion of caspase-1 (Casp1) was analyzed by immunoblotting, and IL-1β or IL-18 secretion was measured by ELISA. ( C ) Secretion of IL-1β secretion was confirmed by immunoblotting. ( D ) For cytotoxicity, BMDMs were treated with the indicated dosages of LNT, and cell number was measured by an automated cell counter. Triton x-100 (1%, Triton) treatment led to cell death. All immunoblot data shown are representative of at least three independent experiments. Bar graph presents the mean ± SD.

Techniques Used: Activation Assay, Enzyme-linked Immunosorbent Assay

37) Product Images from "cGAS drives non-canonical inflammasome activation in age-related macular degeneration"

Article Title: cGAS drives non-canonical inflammasome activation in age-related macular degeneration

Journal: Nature medicine

doi: 10.1038/nm.4450

Gasdermin D in geographic atrophy and RPE degeneration ( a ) Top, fundus photographs of eyes of WT (n = 6 eyes), and Gsdmd −/− (n = 10 eyes) mice subretinally injected with vehicle or Alu RNA. Bottom, immunofluorescence staining of zonula occludens-1 (ZO-1) on RPE flat mounts of the same eyes showing RPE cell boundaries. ( b ) IL-18 secretion by WT and Gsdmd −/− mouse RPE cell s mock transfected or transfected with Alu RNA (n = 3 independent experiments; Data presented are mean ± SD, * P = 0.01, two-tailed t test) ( c ) Immunoblots of pro-caspase-1 (pro-Casp1) and the p10 cleavage product of caspase-1 (Casp1 p10) in WT and Gsdmd −/− mouse RPE cells mock transfected or transfected with Alu RNA. ( d ) Immunoblots of pro-caspase-11 (pro-Casp11) and the p30 cleavage product of caspase-11 (Casp11 p30) in WT and Gsdmd −/− mouse RPE cells mock transfected or transfected with Alu RNA. ( e ) Immunoblots of Gasdermin D and cleavage product of Gasdermin D (Gsdmd p30) in mock transfected or Alu RNA transfected human primary RPE cells, WT mouse primary RPE cells, and WT BMDM as well as in RPE tissue from WT mice subretinally injected with vehicle or Alu RNA. ( f , g ) Fundus photographs and immunofluorescence staining of zonula occludens-1 (ZO-1) on RPE flat mounts, ( f ) Gsdmd −/− mice reconstituted via in vivo subretinal transfection of empty vector plasmid (pNull; n = 4 eyes), plasmids expressing wild type gasdermin D (pGSDMD-WT; n = 4 eyes) or mutant gasdermin D incapable of undergoing p30 cleavage (pGSDMD-D276A; n = 5 eyes) subretinally injected with Alu RNA. ( g ) Gsdmd −/− mice subretinally administered vehicle control (Vehicle; n = 4 eyes), recombinant mature IL-18 (recIL-18; n = 4 eyes), mature IL-18 expression plasmid (pIL-18ss; n = 5 eyes) or empty vector control (pNull; n = 4 eyes) were subretinally injected with Alu RNA. ( h ) RT-qPCR examination of GSDMD mRNA abundance in the RPE tissue of human AMD eyes (n = 7 eyes) and in healthy age-matched control eyes ( n = 6 eyes). * P = 0.045, two-tailed t test; error bars denote geometric means with 95% confidence intervals. ( i ) Immunolocalization of gasdermin D in the RPE of human geographic atrophy eyes and age-matched healthy controls. For all immunoblots, cropped gel image of bands of interest of representative immunoblots of three independent experiments and densitometric analysis (mean (SEM)) are shown. In a , f and g , binary (Healthy %) and morphometric (PM, polymegethism (mean (SEM)) quantification of RPE degeneration are shown (Fisher’s exact test for binary; two-tailed t test for morphometry; * P
Figure Legend Snippet: Gasdermin D in geographic atrophy and RPE degeneration ( a ) Top, fundus photographs of eyes of WT (n = 6 eyes), and Gsdmd −/− (n = 10 eyes) mice subretinally injected with vehicle or Alu RNA. Bottom, immunofluorescence staining of zonula occludens-1 (ZO-1) on RPE flat mounts of the same eyes showing RPE cell boundaries. ( b ) IL-18 secretion by WT and Gsdmd −/− mouse RPE cell s mock transfected or transfected with Alu RNA (n = 3 independent experiments; Data presented are mean ± SD, * P = 0.01, two-tailed t test) ( c ) Immunoblots of pro-caspase-1 (pro-Casp1) and the p10 cleavage product of caspase-1 (Casp1 p10) in WT and Gsdmd −/− mouse RPE cells mock transfected or transfected with Alu RNA. ( d ) Immunoblots of pro-caspase-11 (pro-Casp11) and the p30 cleavage product of caspase-11 (Casp11 p30) in WT and Gsdmd −/− mouse RPE cells mock transfected or transfected with Alu RNA. ( e ) Immunoblots of Gasdermin D and cleavage product of Gasdermin D (Gsdmd p30) in mock transfected or Alu RNA transfected human primary RPE cells, WT mouse primary RPE cells, and WT BMDM as well as in RPE tissue from WT mice subretinally injected with vehicle or Alu RNA. ( f , g ) Fundus photographs and immunofluorescence staining of zonula occludens-1 (ZO-1) on RPE flat mounts, ( f ) Gsdmd −/− mice reconstituted via in vivo subretinal transfection of empty vector plasmid (pNull; n = 4 eyes), plasmids expressing wild type gasdermin D (pGSDMD-WT; n = 4 eyes) or mutant gasdermin D incapable of undergoing p30 cleavage (pGSDMD-D276A; n = 5 eyes) subretinally injected with Alu RNA. ( g ) Gsdmd −/− mice subretinally administered vehicle control (Vehicle; n = 4 eyes), recombinant mature IL-18 (recIL-18; n = 4 eyes), mature IL-18 expression plasmid (pIL-18ss; n = 5 eyes) or empty vector control (pNull; n = 4 eyes) were subretinally injected with Alu RNA. ( h ) RT-qPCR examination of GSDMD mRNA abundance in the RPE tissue of human AMD eyes (n = 7 eyes) and in healthy age-matched control eyes ( n = 6 eyes). * P = 0.045, two-tailed t test; error bars denote geometric means with 95% confidence intervals. ( i ) Immunolocalization of gasdermin D in the RPE of human geographic atrophy eyes and age-matched healthy controls. For all immunoblots, cropped gel image of bands of interest of representative immunoblots of three independent experiments and densitometric analysis (mean (SEM)) are shown. In a , f and g , binary (Healthy %) and morphometric (PM, polymegethism (mean (SEM)) quantification of RPE degeneration are shown (Fisher’s exact test for binary; two-tailed t test for morphometry; * P

Techniques Used: Mouse Assay, Injection, Immunofluorescence, Staining, Transfection, Two Tailed Test, Western Blot, In Vivo, Plasmid Preparation, Expressing, Mutagenesis, Recombinant, Quantitative RT-PCR

cGAS driven signaling licenses non-canonical inflammasome and RPE degeneration ( a ) Relative abundance of Ifnb mRNA in WT and Mb21d1 −/− mouse RPE cells mock-transfected or transfected with Alu RNA. Data presented are mean ± SEM ; n = 4 cell culture replicates; * P = 0.0001, two-tailed t test. ( b ) Immunoblots of pro-caspase-1 (pro-Casp1) and the p20 cleavage product of caspase-1 (Casp1 p20) in WT and Mb21d1 −/− mouse RPE cells transfected with Alu expression plasmid (pAlu) or empty vector control (pNull). ( c ) Immunoblots of pro-caspase-11 (pro-Casp1) and the p30 cleavage product of caspase-1 (Casp11 p30) in WT and Mb21d1 −/− mouse RPE cells transfected with Alu expression plasmid (pAlu) or empty vector control (pNull). ( d ) IL-18 secretion by WT and Mb21d1 −/− mouse RPE cells mock transfected or transfected with Alu RNA. Data presented are mean ± SD ; n = 3 independent experiments; * P = 0.032, two-tailed t test. ( e ) Relative abundance of IFNB mRNA in control (sh Scramble) or cGAS shRNA knockdown human RPE cells transfected with or DICER1 or control (Ctr) anti-sense oligonucleotides (AS). Data presented are mean ± SEM ; n = 3 cell culture replicates; * P = 0.0002, two-tailed t test. ( f ) Immunoblot of phosphorylated STAT2 (pSTAT2); pro-caspase-4 and casp4 p30; pro-caspase-1 and p20 cleavage casp1 p20 in control (sh Scramble) or cGAS shRNA knockdown human RPE cells mock-transfected or transfected with Alu RNA. Knockdown efficiency of cGAS is shown by cGAS immunoblot and tubulin was used as a loading control. ( g ) Fundus photographs and immunofluorescence staining of zonula occludens-1 (ZO-1) on RPE flat mounts of WT (n = 6 eyes) and Mb21d1 −/− (n = 8 eyes) mice subretinally injected with vehicle or Alu RNA. ( h ) Fundus photographs and immunofluorescence staining of zonula occludens-1 (ZO-1) on RPE flat mounts of Mb21d1 −/− (n = 7 eyes) mice reconstituted by in vivo transfection of cGAS expression plasmid (pFlag-cGAS) or control GFP expression plasmid (pFlag-GFP), subretinally injected with Alu RNA. ( i ) Fundus photographs and immunofluorescence staining of zonula occludens-1 (ZO-1) on RPE flat mounts of Mb21d1 −/− mice subretinally co-administered Alu RNA with recombinant IFN-β (n = 6 eyes), vehicle control (n = 6 eyes), IFN-β expression plasmid (pIFNB; n = 5 eyes) or empty vector control (pNull; n = 5 eyes). For all immunoblots, cropped gel image of bands of interest of representative immunoblots of three independent experiments and densitometric analysis (mean (SEM)) are shown. In g , h and i , binary (Healthy %) and morphometric (PM, polymegethism (mean (SEM)) quantification of RPE degeneration are shown (Fisher’s exact test for binary; two-tailed t test for morphometry; * P
Figure Legend Snippet: cGAS driven signaling licenses non-canonical inflammasome and RPE degeneration ( a ) Relative abundance of Ifnb mRNA in WT and Mb21d1 −/− mouse RPE cells mock-transfected or transfected with Alu RNA. Data presented are mean ± SEM ; n = 4 cell culture replicates; * P = 0.0001, two-tailed t test. ( b ) Immunoblots of pro-caspase-1 (pro-Casp1) and the p20 cleavage product of caspase-1 (Casp1 p20) in WT and Mb21d1 −/− mouse RPE cells transfected with Alu expression plasmid (pAlu) or empty vector control (pNull). ( c ) Immunoblots of pro-caspase-11 (pro-Casp1) and the p30 cleavage product of caspase-1 (Casp11 p30) in WT and Mb21d1 −/− mouse RPE cells transfected with Alu expression plasmid (pAlu) or empty vector control (pNull). ( d ) IL-18 secretion by WT and Mb21d1 −/− mouse RPE cells mock transfected or transfected with Alu RNA. Data presented are mean ± SD ; n = 3 independent experiments; * P = 0.032, two-tailed t test. ( e ) Relative abundance of IFNB mRNA in control (sh Scramble) or cGAS shRNA knockdown human RPE cells transfected with or DICER1 or control (Ctr) anti-sense oligonucleotides (AS). Data presented are mean ± SEM ; n = 3 cell culture replicates; * P = 0.0002, two-tailed t test. ( f ) Immunoblot of phosphorylated STAT2 (pSTAT2); pro-caspase-4 and casp4 p30; pro-caspase-1 and p20 cleavage casp1 p20 in control (sh Scramble) or cGAS shRNA knockdown human RPE cells mock-transfected or transfected with Alu RNA. Knockdown efficiency of cGAS is shown by cGAS immunoblot and tubulin was used as a loading control. ( g ) Fundus photographs and immunofluorescence staining of zonula occludens-1 (ZO-1) on RPE flat mounts of WT (n = 6 eyes) and Mb21d1 −/− (n = 8 eyes) mice subretinally injected with vehicle or Alu RNA. ( h ) Fundus photographs and immunofluorescence staining of zonula occludens-1 (ZO-1) on RPE flat mounts of Mb21d1 −/− (n = 7 eyes) mice reconstituted by in vivo transfection of cGAS expression plasmid (pFlag-cGAS) or control GFP expression plasmid (pFlag-GFP), subretinally injected with Alu RNA. ( i ) Fundus photographs and immunofluorescence staining of zonula occludens-1 (ZO-1) on RPE flat mounts of Mb21d1 −/− mice subretinally co-administered Alu RNA with recombinant IFN-β (n = 6 eyes), vehicle control (n = 6 eyes), IFN-β expression plasmid (pIFNB; n = 5 eyes) or empty vector control (pNull; n = 5 eyes). For all immunoblots, cropped gel image of bands of interest of representative immunoblots of three independent experiments and densitometric analysis (mean (SEM)) are shown. In g , h and i , binary (Healthy %) and morphometric (PM, polymegethism (mean (SEM)) quantification of RPE degeneration are shown (Fisher’s exact test for binary; two-tailed t test for morphometry; * P

Techniques Used: Transfection, Cell Culture, Two Tailed Test, Western Blot, Expressing, Plasmid Preparation, shRNA, Immunofluorescence, Staining, Mouse Assay, Injection, In Vivo, Recombinant

mtDNA in non-canonical inflammasome activation and RPE degeneration ( a ) Relative abundance of cytosolic mitochondrial DNA (mtDNA) in human RPE cells mock-transfected or transfected with Alu RNA (Data presented are mean ± SEM; n = 3 independent experiments; * P = 0.0018, two-tailed t test). ( b ) Relative enrichment of mtDNA in cGAS immunoprecipitate in ChIP like pull-down assay. Mock or Alu RNA transfected, indicated mouse embryonic fibroblast (MEF) were analyzed upon HA-cGAS immunoprecipitation with anti-HA antibody or isotype conrol. Data presented are mean ± SEM; n = 3; * P = 0.008, two-tailed t test. ( c ) Relative abundance of cytosolic mitochondrial DNA (mtDNA) in WT and Ppif −/− mouse RPE cells mock transfected (n = 4 cell culture replicates) or transfected with Alu RNA (Data presented are mean ± SEM; n = 6 cell culture replicates; * P = 0.004, two-tailed t test). ( d ) Fundus photographs and immunofluorescence staining of zonula occludens-1 (ZO-1) on RPE flat mounts of WT (n = 6 eyes) and Ppif −/− (n = 12 eyes) mice subretinally injected with vehicle or Alu RNA. ( e ) Immunoblot for procaspase-1 (procasp-1) and p20 cleavage product of caspase-1 in WT and Ppif −/− mouse RPE cells mock transfected or transfected with Alu RNA. ( f ) Immunoblot for procaspase-11 (procasp-11) and p30 cleavage product of caspase-11 (Casp11 p30) in WT and Ppif −/− mouse RPE cells mock transfected or transfected with Alu RNA. ( g ) Immunoblot for procaspase-4 (procasp-4) and p30 cleavage product of caspase-4 (Casp4 p30) in WT and mitochondrial DNA deficient Rho 0 ARPE19 human RPE cells mock-transfected or transfected with Alu RNA. ( h , i ) WT and mitochondrial DNA deficient Rho 0 ARPE19 human RPE cells mock-transfected or transfected with Alu RNA. (h) IL-18 secretion; data presented are mean ± SD; n = 4 independent experiments; * P = 0.0001, two-tailed t test. ( i ) IFN- β secretion; data presented are mean ± SD; n = 4 independent experiments; * P = 0.004, two-tailed t test. ( j ) Fundus photographs and immunofluorescence staining of zonula occludens-1 (ZO-1) on RPE flat mounts of the Ppif −/− mice subretinally co-administered Alu RNA with recombinant IFN-β (n = 5 eyes) or vehicle control (n = 5 eyes); or IFN-β expression plasmid (pIFNB; n = 6 eyes) or empty vector control (pNull; n = 5 eyes). For all immunoblots, cropped gel image of bands of interest of representative immunoblots of three independent experiments and densitometric analysis (mean (SEM)) are shown. In d and j , binary (Healthy %) and morphometric (PM, polymegethism (mean (SEM)) quantification of RPE degeneration are shown (Fisher’s exact test for binary; two-tailed t test for morphometry; * P
Figure Legend Snippet: mtDNA in non-canonical inflammasome activation and RPE degeneration ( a ) Relative abundance of cytosolic mitochondrial DNA (mtDNA) in human RPE cells mock-transfected or transfected with Alu RNA (Data presented are mean ± SEM; n = 3 independent experiments; * P = 0.0018, two-tailed t test). ( b ) Relative enrichment of mtDNA in cGAS immunoprecipitate in ChIP like pull-down assay. Mock or Alu RNA transfected, indicated mouse embryonic fibroblast (MEF) were analyzed upon HA-cGAS immunoprecipitation with anti-HA antibody or isotype conrol. Data presented are mean ± SEM; n = 3; * P = 0.008, two-tailed t test. ( c ) Relative abundance of cytosolic mitochondrial DNA (mtDNA) in WT and Ppif −/− mouse RPE cells mock transfected (n = 4 cell culture replicates) or transfected with Alu RNA (Data presented are mean ± SEM; n = 6 cell culture replicates; * P = 0.004, two-tailed t test). ( d ) Fundus photographs and immunofluorescence staining of zonula occludens-1 (ZO-1) on RPE flat mounts of WT (n = 6 eyes) and Ppif −/− (n = 12 eyes) mice subretinally injected with vehicle or Alu RNA. ( e ) Immunoblot for procaspase-1 (procasp-1) and p20 cleavage product of caspase-1 in WT and Ppif −/− mouse RPE cells mock transfected or transfected with Alu RNA. ( f ) Immunoblot for procaspase-11 (procasp-11) and p30 cleavage product of caspase-11 (Casp11 p30) in WT and Ppif −/− mouse RPE cells mock transfected or transfected with Alu RNA. ( g ) Immunoblot for procaspase-4 (procasp-4) and p30 cleavage product of caspase-4 (Casp4 p30) in WT and mitochondrial DNA deficient Rho 0 ARPE19 human RPE cells mock-transfected or transfected with Alu RNA. ( h , i ) WT and mitochondrial DNA deficient Rho 0 ARPE19 human RPE cells mock-transfected or transfected with Alu RNA. (h) IL-18 secretion; data presented are mean ± SD; n = 4 independent experiments; * P = 0.0001, two-tailed t test. ( i ) IFN- β secretion; data presented are mean ± SD; n = 4 independent experiments; * P = 0.004, two-tailed t test. ( j ) Fundus photographs and immunofluorescence staining of zonula occludens-1 (ZO-1) on RPE flat mounts of the Ppif −/− mice subretinally co-administered Alu RNA with recombinant IFN-β (n = 5 eyes) or vehicle control (n = 5 eyes); or IFN-β expression plasmid (pIFNB; n = 6 eyes) or empty vector control (pNull; n = 5 eyes). For all immunoblots, cropped gel image of bands of interest of representative immunoblots of three independent experiments and densitometric analysis (mean (SEM)) are shown. In d and j , binary (Healthy %) and morphometric (PM, polymegethism (mean (SEM)) quantification of RPE degeneration are shown (Fisher’s exact test for binary; two-tailed t test for morphometry; * P

Techniques Used: Activation Assay, Transfection, Two Tailed Test, Chromatin Immunoprecipitation, Pull Down Assay, Immunoprecipitation, Cell Culture, Immunofluorescence, Staining, Mouse Assay, Injection, Recombinant, Expressing, Plasmid Preparation, Western Blot

Caspase-4/11 in geographic atrophy and RPE degeneration ( a ) Left and top quadrants, immunoblots for pro-caspase-4 (pro-Casp4) and the p30 cleavage product of caspase-4 (Casp4 p30) in the RPE of human eyes with geographic atrophy (dry AMD) as compared to unaffected controls (Ctr). Specific bands of interest are indicated by arrowheads. Lower right quadrant, densitometry of the bands corresponding to caspase-4 p30 normalized to loading control. The molecular weight markers are indicated on the left side of the blot (Data are presented as mean ± SD; n = 3 control eyes; n = 6 dry AMD eyes; *P = 0.002, two-tailed t test). ( b ) Immunoblots for pro-Casp4 and Casp4 p30 in human RPE cells mock transfected (just transfection mixture) or transfected with Alu RNA; Alu expression plasmid (pAlu) or empty vector (pNull); or DICER1 or control (Ctr) anti-sense oligonucleotides (AS). Specific bands of interest are indicated by arrowheads. ( c ) Immunoblot for pro-caspase-11 (Pro-Casp11) and the p30 cleavage product of caspase-11 (Casp11 p30) in RPE tissue of WT mice injected subretinally with Alu RNA or vehicle (Vehi). n = 3 mice per group. (d,e) Top, fundus photographs of the retinas of WT (n = 8 eyes) and Casp11 −/− (n = 10 eyes) mice, ( d ) and Casp11 −/− (n = 8 eyes) mice expressing a human caspase-4 transgene ( Casp11 −/− hCasp4 Tg ) ( e ) injected with vehicle or Alu RNA. The degenerated retinal area is outlined by blue arrowheads. Bottom, immunostaining with zonula occludens-1 (ZO-1) antibody to visualize RPE cellular boundaries; loss of regular hexagonal cellular boundaries is indicative of degenerated RPE. ( f ) Immunoblots of pro-caspase-1 (pro-Casp1) and the p20 cleavage product of caspase-1 (Casp1 p20) in RPE tissue of WT and Casp11 −/− mice injected subretinally with vehicle (Vehi) or Alu RNA. n = 3 mice per group. ( g ) Immunoblots of pro-caspase-1 and the p20 cleavage product of caspase-1 in WT and Casp11 −/− mouse RPE cells treated with Alu RNA. ( h ) IL-18 secretion by WT and Casp11 −/− mouse RPE cell s mock transfected or transfected with Alu RNA. n = 3 independent experiments. Data presented are mean ± SD; * P = 0.014, two-tailed t test. ( i ) Top, fundus photographs of the retinas of WT (n = 8 eyes), caspase-1 and caspase-11 deficient (n = 7 eyes) mice ( Casp1 −/− Casp11 129mt/129mt ) as well as Casp1 −/− Casp11 129mt/129mt (n = 8 eyes) mice expressing functional mouse caspase-11 from a bacterial artificial chromosome transgene ( Casp1 −/− Casp11 129mt/129mt Casp11 Tg ) subretinally injected with vehicle or Alu RNA. For all immunoblots, cropped gel image of bands of interest of representative immunoblots of three independent experiments and densitometric analysis (mean (SEM)) are shown. Tubulin or β-actin or Vinculin was as a loading control as indicated in each blot. In d , e and i , binary (Healthy %) and morphometric quantification (PM, polymegethism (mean (SEM))) of RPE degeneration are shown (Fisher’s exact test for binary; two-tailed t test for morphometry; * P
Figure Legend Snippet: Caspase-4/11 in geographic atrophy and RPE degeneration ( a ) Left and top quadrants, immunoblots for pro-caspase-4 (pro-Casp4) and the p30 cleavage product of caspase-4 (Casp4 p30) in the RPE of human eyes with geographic atrophy (dry AMD) as compared to unaffected controls (Ctr). Specific bands of interest are indicated by arrowheads. Lower right quadrant, densitometry of the bands corresponding to caspase-4 p30 normalized to loading control. The molecular weight markers are indicated on the left side of the blot (Data are presented as mean ± SD; n = 3 control eyes; n = 6 dry AMD eyes; *P = 0.002, two-tailed t test). ( b ) Immunoblots for pro-Casp4 and Casp4 p30 in human RPE cells mock transfected (just transfection mixture) or transfected with Alu RNA; Alu expression plasmid (pAlu) or empty vector (pNull); or DICER1 or control (Ctr) anti-sense oligonucleotides (AS). Specific bands of interest are indicated by arrowheads. ( c ) Immunoblot for pro-caspase-11 (Pro-Casp11) and the p30 cleavage product of caspase-11 (Casp11 p30) in RPE tissue of WT mice injected subretinally with Alu RNA or vehicle (Vehi). n = 3 mice per group. (d,e) Top, fundus photographs of the retinas of WT (n = 8 eyes) and Casp11 −/− (n = 10 eyes) mice, ( d ) and Casp11 −/− (n = 8 eyes) mice expressing a human caspase-4 transgene ( Casp11 −/− hCasp4 Tg ) ( e ) injected with vehicle or Alu RNA. The degenerated retinal area is outlined by blue arrowheads. Bottom, immunostaining with zonula occludens-1 (ZO-1) antibody to visualize RPE cellular boundaries; loss of regular hexagonal cellular boundaries is indicative of degenerated RPE. ( f ) Immunoblots of pro-caspase-1 (pro-Casp1) and the p20 cleavage product of caspase-1 (Casp1 p20) in RPE tissue of WT and Casp11 −/− mice injected subretinally with vehicle (Vehi) or Alu RNA. n = 3 mice per group. ( g ) Immunoblots of pro-caspase-1 and the p20 cleavage product of caspase-1 in WT and Casp11 −/− mouse RPE cells treated with Alu RNA. ( h ) IL-18 secretion by WT and Casp11 −/− mouse RPE cell s mock transfected or transfected with Alu RNA. n = 3 independent experiments. Data presented are mean ± SD; * P = 0.014, two-tailed t test. ( i ) Top, fundus photographs of the retinas of WT (n = 8 eyes), caspase-1 and caspase-11 deficient (n = 7 eyes) mice ( Casp1 −/− Casp11 129mt/129mt ) as well as Casp1 −/− Casp11 129mt/129mt (n = 8 eyes) mice expressing functional mouse caspase-11 from a bacterial artificial chromosome transgene ( Casp1 −/− Casp11 129mt/129mt Casp11 Tg ) subretinally injected with vehicle or Alu RNA. For all immunoblots, cropped gel image of bands of interest of representative immunoblots of three independent experiments and densitometric analysis (mean (SEM)) are shown. Tubulin or β-actin or Vinculin was as a loading control as indicated in each blot. In d , e and i , binary (Healthy %) and morphometric quantification (PM, polymegethism (mean (SEM))) of RPE degeneration are shown (Fisher’s exact test for binary; two-tailed t test for morphometry; * P

Techniques Used: Western Blot, Molecular Weight, Two Tailed Test, Transfection, Expressing, Plasmid Preparation, Mouse Assay, Injection, Immunostaining, Functional Assay

38) Product Images from "GM-CSF Signalling Boosts Dramatically IL-1Production"

Article Title: GM-CSF Signalling Boosts Dramatically IL-1Production

Journal: PLoS ONE

doi: 10.1371/journal.pone.0023025

GM-CSF boosts LPS-induced IL-1 secretion. (A) CD11b + fraction of FLT3L derived DCs was stimulated 24 h with a wide range of different LPS concentrations (0.001–10 µg/ml) in absence (white circles) or presence of 5 ng/ml GM-CSF (black circles). 5 mM ATP was added as a danger signal. Released IL-1β, IL-1α, TNF-α and IL-6 were measured in the culture supernatants by standard ELISA and each value represents the mean of triplicates +/− SD. (B) CD11b + fraction of FLT3L generated DCs was primed for 24 h with 100 ng/ml LPS with (back bars) or without (white bars) 5 ng/ml GM-CSF and stimulated with different danger signals (5 mM ATP, 1 µM nigericin, 100 µg/ml MSU, 200 µg/ml Alu). Each bar represents the mean of triplicates +/− SD. (C) CD11b + fraction of FLT3L generated DCs was primed with TLR agonists (100 ng/ml LPS and Pam 3 CSK 4 ), Dectin agonist, Curdlan (100 µg/ml) and pro-inflammatory cytokine TNF-α (100 ng/ml) in absence (white bars) or presence (back bars) of 5 ng/ml GM-CSF and stimulated subsequently with ATP. Each bar represents the mean of triplicates +/− SD. (D) GM-CSF derived BM DCs, M-CSF-derived BM MØ as well as L929-derived BM MØ were compared to the CD11b + fraction of FLT3L-derived DCs for their capacity to secrete IL-1β upon 24 h LPS stimulation (100 ng/ml) in absence or in presence of GM-CSF (5 ng/ml). ATP was added as danger signal. Both, WT (black bars) and GM-CSF R−/− cells (white bars) were tested. Each bar represents the mean of triplicates +/− SD. All results are representative of at least two independent experiments.
Figure Legend Snippet: GM-CSF boosts LPS-induced IL-1 secretion. (A) CD11b + fraction of FLT3L derived DCs was stimulated 24 h with a wide range of different LPS concentrations (0.001–10 µg/ml) in absence (white circles) or presence of 5 ng/ml GM-CSF (black circles). 5 mM ATP was added as a danger signal. Released IL-1β, IL-1α, TNF-α and IL-6 were measured in the culture supernatants by standard ELISA and each value represents the mean of triplicates +/− SD. (B) CD11b + fraction of FLT3L generated DCs was primed for 24 h with 100 ng/ml LPS with (back bars) or without (white bars) 5 ng/ml GM-CSF and stimulated with different danger signals (5 mM ATP, 1 µM nigericin, 100 µg/ml MSU, 200 µg/ml Alu). Each bar represents the mean of triplicates +/− SD. (C) CD11b + fraction of FLT3L generated DCs was primed with TLR agonists (100 ng/ml LPS and Pam 3 CSK 4 ), Dectin agonist, Curdlan (100 µg/ml) and pro-inflammatory cytokine TNF-α (100 ng/ml) in absence (white bars) or presence (back bars) of 5 ng/ml GM-CSF and stimulated subsequently with ATP. Each bar represents the mean of triplicates +/− SD. (D) GM-CSF derived BM DCs, M-CSF-derived BM MØ as well as L929-derived BM MØ were compared to the CD11b + fraction of FLT3L-derived DCs for their capacity to secrete IL-1β upon 24 h LPS stimulation (100 ng/ml) in absence or in presence of GM-CSF (5 ng/ml). ATP was added as danger signal. Both, WT (black bars) and GM-CSF R−/− cells (white bars) were tested. Each bar represents the mean of triplicates +/− SD. All results are representative of at least two independent experiments.

Techniques Used: Derivative Assay, Enzyme-linked Immunosorbent Assay, Generated

LPS and LPS/GM-CSF induce equal cleavage of caspase-1 in presence of a danger signal. Measurement of bioactive secreted IL-1β p17 and active caspase p20 in serum free culture supernatants of WT and GM-CSFR−/− L929-derived BM MØ treated overnight with 100 ng/ml LPS, 5 ng/ml GM-CSF or a combination of both and then pulsed for 1 h with Nigericin. Upper panel shows the quantification of the amount of IL-1β released measured by ELISA. Results are representative of two independent experiments.
Figure Legend Snippet: LPS and LPS/GM-CSF induce equal cleavage of caspase-1 in presence of a danger signal. Measurement of bioactive secreted IL-1β p17 and active caspase p20 in serum free culture supernatants of WT and GM-CSFR−/− L929-derived BM MØ treated overnight with 100 ng/ml LPS, 5 ng/ml GM-CSF or a combination of both and then pulsed for 1 h with Nigericin. Upper panel shows the quantification of the amount of IL-1β released measured by ELISA. Results are representative of two independent experiments.

Techniques Used: Derivative Assay, Enzyme-linked Immunosorbent Assay

39) Product Images from "Inflammasome activation plays an important role in the development of spontaneous colitis"

Article Title: Inflammasome activation plays an important role in the development of spontaneous colitis

Journal: Mucosal immunology

doi: 10.1038/mi.2014.1

Inhibition of inflammasome activities leads to reduced IL-17 production from MLN cells of IL-10 KO mice ( a ) and ( b ) MLN cells from WT and IL-10 KO mice were stimulated with anti-CD3 antibody in combination with LPS (100 ng/ml) or recombinant IL-1β (25 ng/ml) as indicated for 48 hours. The production of IL-17 was measured by ELISA. ( c ) and ( d ) MLN cells from IL-10 deficient mice were treated with anti-CD3 antibody or LPS in the presence of IL-1R antagonist (IL-1Ra) or caspase 1 inhibitor Z-VAD-FMK (ZVAD), and IL-17 production was measured 48 hours after treatment. Results are reported as mean ± SD of the pool of two experiments (5–6 mice per group). ** p
Figure Legend Snippet: Inhibition of inflammasome activities leads to reduced IL-17 production from MLN cells of IL-10 KO mice ( a ) and ( b ) MLN cells from WT and IL-10 KO mice were stimulated with anti-CD3 antibody in combination with LPS (100 ng/ml) or recombinant IL-1β (25 ng/ml) as indicated for 48 hours. The production of IL-17 was measured by ELISA. ( c ) and ( d ) MLN cells from IL-10 deficient mice were treated with anti-CD3 antibody or LPS in the presence of IL-1R antagonist (IL-1Ra) or caspase 1 inhibitor Z-VAD-FMK (ZVAD), and IL-17 production was measured 48 hours after treatment. Results are reported as mean ± SD of the pool of two experiments (5–6 mice per group). ** p

Techniques Used: Inhibition, Mouse Assay, Recombinant, Enzyme-linked Immunosorbent Assay

40) Product Images from "MicroRNA-488 and -920 regulate the production of proinflammatory cytokines in acute gouty arthritis"

Article Title: MicroRNA-488 and -920 regulate the production of proinflammatory cytokines in acute gouty arthritis

Journal: Arthritis Research & Therapy

doi: 10.1186/s13075-017-1418-6

MicroRNA (miR, miRNA)-488 and miR-920 suppress monosodium urate (MSU)-induced expression of proinflammatory cytokines in THP-1 cells. The miRNA mimics or negative control (NC) mimics (50 nM) were transfected into THP-1 cells using Lipofectamine RNAiMAX reagent in accordance with the manufacturer’s instructions. After 24 h of transfection, cells were stimulated for 3 h with 0.5 μM 12-myristate 13-acetate. Then, cells were washed and stimulated with 250 μg/ml MSU crystals for 24 h to detect the production of proinflammatory cytokines. After the treatment, the cells were collected and analyzed by quantitative real-time polymerase chain reaction ( a , c ). The cell culture supernatants were also collected to detect the concentrations of interleukin (IL)-8 and tumor necrosis factor (TNF)-α by enzyme-linked immunosorbent assay ( b , d ). Values are expressed as mean ± SEM of three independent experiments, each of which was run in triplicate. # P
Figure Legend Snippet: MicroRNA (miR, miRNA)-488 and miR-920 suppress monosodium urate (MSU)-induced expression of proinflammatory cytokines in THP-1 cells. The miRNA mimics or negative control (NC) mimics (50 nM) were transfected into THP-1 cells using Lipofectamine RNAiMAX reagent in accordance with the manufacturer’s instructions. After 24 h of transfection, cells were stimulated for 3 h with 0.5 μM 12-myristate 13-acetate. Then, cells were washed and stimulated with 250 μg/ml MSU crystals for 24 h to detect the production of proinflammatory cytokines. After the treatment, the cells were collected and analyzed by quantitative real-time polymerase chain reaction ( a , c ). The cell culture supernatants were also collected to detect the concentrations of interleukin (IL)-8 and tumor necrosis factor (TNF)-α by enzyme-linked immunosorbent assay ( b , d ). Values are expressed as mean ± SEM of three independent experiments, each of which was run in triplicate. # P

Techniques Used: Expressing, Negative Control, Transfection, Real-time Polymerase Chain Reaction, Cell Culture, Enzyme-linked Immunosorbent Assay

Monosodium urate (MSU) crystals promote the expression of proinflammatory cytokines in THP-1 cells. THP-1 cells were stimulated by the indicated concentration of MSU crystals. The messenger (mRNA) expression of interleukin (IL)-1β, IL-8, and tumor necrosis factor (TNF)-α were detected by quantitative real-time polymerase chain reaction ( a – c ). Protein expression of IL-1β, IL-8, and TNF-α was detected by enzyme-linked immunosorbent assay ( d – f ). Values are expressed as mean ± SEM of three independent experiments, each of which was run in triplicate. * P
Figure Legend Snippet: Monosodium urate (MSU) crystals promote the expression of proinflammatory cytokines in THP-1 cells. THP-1 cells were stimulated by the indicated concentration of MSU crystals. The messenger (mRNA) expression of interleukin (IL)-1β, IL-8, and tumor necrosis factor (TNF)-α were detected by quantitative real-time polymerase chain reaction ( a – c ). Protein expression of IL-1β, IL-8, and TNF-α was detected by enzyme-linked immunosorbent assay ( d – f ). Values are expressed as mean ± SEM of three independent experiments, each of which was run in triplicate. * P

Techniques Used: Expressing, Concentration Assay, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

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Article Title: Recombinant human proteoglycan-4 reduces phagocytosis of urate crystals and downstream nuclear factor kappa B and inflammasome activation and production of cytokines and chemokines in human and murine macrophages
Article Snippet: .. THP-1 macrophages were treated with endotoxin-free MSU crystals (100μg/ml; Invivogen, USA) ± bovine submaxillary mucin (BSM; molecular mass > 1000 KDa) (Sigma Aldrich) (25 μg/ml) or rhPRG4 (molecular mass is approximately 240 KDa) (100 μg/ml) for 2 and 4 h at 37 °C. rhPRG4 is an endotoxin-free full-length product produced by CHO-M cells (Lubris, Framingham, MA, USA) [ ]. ..

Marker:

Article Title: Uric acid crystals induces placental inflammation and alters trophoblast function via an IL-1-dependent pathway: implication for FGR
Article Snippet: .. Both MSU crystals and IL-1β significantly induced cell death in cytotrophoblast cultures, as seen by the 3.3-fold and 2.7-fold increase, respectively, in the percentage of cells positive for the apoptotic marker M30 at 48h ( ). .. Treatment of MSU crystals-exposed cytotrophoblasts with IL-1Ra was protective, with decreased percentage of M30+ apoptotic cells ( ).

Injection:

Article Title: The Immunomodulatory Metabolite Itaconate Modifies NLRP3 and Inhibits Inflammasome Activation
Article Snippet: .. MSU-Induced Peritonitis Model 6-week old female C57BL/6J mice were injected intraperitoneally with a mixture of 4-OI (50 mg/kg) in 60% cyclodextrin in PBS and MSU crystals (30mg/kg, Invivogen) suspended in PBS for 6 h. Mice were euthanized in a CO2 chamber and peritoneal lavage was performed using 2.5 mL PBS. .. The cells in the lavage fluid were pelleted and the supernatant was removed and analyzed by ELISA for IL-1β and IL-6 concentration.

other:

Article Title: Uric acid crystals induces placental inflammation and alters trophoblast function via an IL-1-dependent pathway: implication for FGR
Article Snippet: Since IL-1β is a well-known mediator of MSU crystals actions in immune cells, and it was strongly induced in term cytotrophoblasts in response to MSU crystals, we addressed the mechanisms of IL-1β production.

Article Title: Uric acid crystals induces placental inflammation and alters trophoblast function via an IL-1-dependent pathway: implication for FGR
Article Snippet: Consistent with these findings in cytotrophoblast, treatment with MSU crystals or IL-1β induced apoptosis in term placental explants, ( ) which was mainly observed in cytotrophoblast cells (arrowheads in ).

Article Title: Uric acid crystals induces placental inflammation and alters trophoblast function via an IL-1-dependent pathway: implication for FGR
Article Snippet: This was predominantly observed within the junctional zone, where all doses of MSU crystals led to a significant elevation of monocytes/macrophages (CD68+ cells) and within the labyrinth (fetal side), where only the highest dose of MSU crystals induced a significant increased in CD68+ monocytes/macrophages ( ).

Article Title: MicroRNA-488 and -920 regulate the production of proinflammatory cytokines in acute gouty arthritis
Article Snippet: MSU crystals can induce a variety of cytokines, including IL-1β, IL-6, IL-8, and TNF-α [ ].

Article Title: Uric acid crystals induces placental inflammation and alters trophoblast function via an IL-1-dependent pathway: implication for FGR
Article Snippet: The secretion of IL-1β induced by MSU crystals in cytotrophoblasts was dependent on the common processor of pro-IL-1β, caspase-1, which was ascertained using a caspase-1 inhibitor ( ).

Mouse Assay:

Article Title: The Immunomodulatory Metabolite Itaconate Modifies NLRP3 and Inhibits Inflammasome Activation
Article Snippet: .. MSU-Induced Peritonitis Model 6-week old female C57BL/6J mice were injected intraperitoneally with a mixture of 4-OI (50 mg/kg) in 60% cyclodextrin in PBS and MSU crystals (30mg/kg, Invivogen) suspended in PBS for 6 h. Mice were euthanized in a CO2 chamber and peritoneal lavage was performed using 2.5 mL PBS. .. The cells in the lavage fluid were pelleted and the supernatant was removed and analyzed by ELISA for IL-1β and IL-6 concentration.

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    InvivoGen msu crystals
    4-OI Reduces Inflammation in a Murine In Vivo Model of Peritonitis and Blocks NLRP3 Inflammasome Activation in Healthy Human and CAPS PBMCs (A–C) IL-1β concentration (A), IL-6 concentration (B), and neutrophil number (C) in the peritoneal lavage fluid of mice injected for 6 h with <t>MSU</t> crystals (30 mg/kg) ± 4-OI (50 mg/kg) (n = 3 for <t>PBS</t> groups, n = 8 for MSU groups). (D) LPS or Pam3CSK4 (14 h) and nigericin (2 h) induced IL-1β release (n = 5 for LPS + nigericin, n = 3 for Pam3CSK4 + nigericin) ± 4-OI or 4-O-2-MS (both 250 μM) from healthy human PBMCs. (E and F) Immunoblot analysis (E) and quantification by densitometry (F, n = 3) of pro- and mature IL-1β protein in lysates and supernatants of human PBMCs treated with LPS (14 h) and nigericin (2 h) ± 4-OI (250 μM). (G) LPS (1 h) induced IL-1β release (n = 3) ± 4-OI (250 μM) or MCC950 (500 nM) from PBMCs isolated from CAPS patients. ∗ p
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    4-OI Reduces Inflammation in a Murine In Vivo Model of Peritonitis and Blocks NLRP3 Inflammasome Activation in Healthy Human and CAPS PBMCs (A–C) IL-1β concentration (A), IL-6 concentration (B), and neutrophil number (C) in the peritoneal lavage fluid of mice injected for 6 h with MSU crystals (30 mg/kg) ± 4-OI (50 mg/kg) (n = 3 for PBS groups, n = 8 for MSU groups). (D) LPS or Pam3CSK4 (14 h) and nigericin (2 h) induced IL-1β release (n = 5 for LPS + nigericin, n = 3 for Pam3CSK4 + nigericin) ± 4-OI or 4-O-2-MS (both 250 μM) from healthy human PBMCs. (E and F) Immunoblot analysis (E) and quantification by densitometry (F, n = 3) of pro- and mature IL-1β protein in lysates and supernatants of human PBMCs treated with LPS (14 h) and nigericin (2 h) ± 4-OI (250 μM). (G) LPS (1 h) induced IL-1β release (n = 3) ± 4-OI (250 μM) or MCC950 (500 nM) from PBMCs isolated from CAPS patients. ∗ p

    Journal: Cell Metabolism

    Article Title: The Immunomodulatory Metabolite Itaconate Modifies NLRP3 and Inhibits Inflammasome Activation

    doi: 10.1016/j.cmet.2020.07.016

    Figure Lengend Snippet: 4-OI Reduces Inflammation in a Murine In Vivo Model of Peritonitis and Blocks NLRP3 Inflammasome Activation in Healthy Human and CAPS PBMCs (A–C) IL-1β concentration (A), IL-6 concentration (B), and neutrophil number (C) in the peritoneal lavage fluid of mice injected for 6 h with MSU crystals (30 mg/kg) ± 4-OI (50 mg/kg) (n = 3 for PBS groups, n = 8 for MSU groups). (D) LPS or Pam3CSK4 (14 h) and nigericin (2 h) induced IL-1β release (n = 5 for LPS + nigericin, n = 3 for Pam3CSK4 + nigericin) ± 4-OI or 4-O-2-MS (both 250 μM) from healthy human PBMCs. (E and F) Immunoblot analysis (E) and quantification by densitometry (F, n = 3) of pro- and mature IL-1β protein in lysates and supernatants of human PBMCs treated with LPS (14 h) and nigericin (2 h) ± 4-OI (250 μM). (G) LPS (1 h) induced IL-1β release (n = 3) ± 4-OI (250 μM) or MCC950 (500 nM) from PBMCs isolated from CAPS patients. ∗ p

    Article Snippet: MSU-Induced Peritonitis Model 6-week old female C57BL/6J mice were injected intraperitoneally with a mixture of 4-OI (50 mg/kg) in 60% cyclodextrin in PBS and MSU crystals (30mg/kg, Invivogen) suspended in PBS for 6 h. Mice were euthanized in a CO2 chamber and peritoneal lavage was performed using 2.5 mL PBS.

    Techniques: In Vivo, Activation Assay, Concentration Assay, Mouse Assay, Injection, Isolation

    MicroRNA (miR, miRNA)-488 and miR-920 suppress monosodium urate (MSU)-induced expression of proinflammatory cytokines in THP-1 cells. The miRNA mimics or negative control (NC) mimics (50 nM) were transfected into THP-1 cells using Lipofectamine RNAiMAX reagent in accordance with the manufacturer’s instructions. After 24 h of transfection, cells were stimulated for 3 h with 0.5 μM 12-myristate 13-acetate. Then, cells were washed and stimulated with 250 μg/ml MSU crystals for 24 h to detect the production of proinflammatory cytokines. After the treatment, the cells were collected and analyzed by quantitative real-time polymerase chain reaction ( a , c ). The cell culture supernatants were also collected to detect the concentrations of interleukin (IL)-8 and tumor necrosis factor (TNF)-α by enzyme-linked immunosorbent assay ( b , d ). Values are expressed as mean ± SEM of three independent experiments, each of which was run in triplicate. # P

    Journal: Arthritis Research & Therapy

    Article Title: MicroRNA-488 and -920 regulate the production of proinflammatory cytokines in acute gouty arthritis

    doi: 10.1186/s13075-017-1418-6

    Figure Lengend Snippet: MicroRNA (miR, miRNA)-488 and miR-920 suppress monosodium urate (MSU)-induced expression of proinflammatory cytokines in THP-1 cells. The miRNA mimics or negative control (NC) mimics (50 nM) were transfected into THP-1 cells using Lipofectamine RNAiMAX reagent in accordance with the manufacturer’s instructions. After 24 h of transfection, cells were stimulated for 3 h with 0.5 μM 12-myristate 13-acetate. Then, cells were washed and stimulated with 250 μg/ml MSU crystals for 24 h to detect the production of proinflammatory cytokines. After the treatment, the cells were collected and analyzed by quantitative real-time polymerase chain reaction ( a , c ). The cell culture supernatants were also collected to detect the concentrations of interleukin (IL)-8 and tumor necrosis factor (TNF)-α by enzyme-linked immunosorbent assay ( b , d ). Values are expressed as mean ± SEM of three independent experiments, each of which was run in triplicate. # P

    Article Snippet: MSU crystals can induce a variety of cytokines, including IL-1β, IL-6, IL-8, and TNF-α [ ].

    Techniques: Expressing, Negative Control, Transfection, Real-time Polymerase Chain Reaction, Cell Culture, Enzyme-linked Immunosorbent Assay

    Monosodium urate (MSU) crystals promote the expression of proinflammatory cytokines in THP-1 cells. THP-1 cells were stimulated by the indicated concentration of MSU crystals. The messenger (mRNA) expression of interleukin (IL)-1β, IL-8, and tumor necrosis factor (TNF)-α were detected by quantitative real-time polymerase chain reaction ( a – c ). Protein expression of IL-1β, IL-8, and TNF-α was detected by enzyme-linked immunosorbent assay ( d – f ). Values are expressed as mean ± SEM of three independent experiments, each of which was run in triplicate. * P

    Journal: Arthritis Research & Therapy

    Article Title: MicroRNA-488 and -920 regulate the production of proinflammatory cytokines in acute gouty arthritis

    doi: 10.1186/s13075-017-1418-6

    Figure Lengend Snippet: Monosodium urate (MSU) crystals promote the expression of proinflammatory cytokines in THP-1 cells. THP-1 cells were stimulated by the indicated concentration of MSU crystals. The messenger (mRNA) expression of interleukin (IL)-1β, IL-8, and tumor necrosis factor (TNF)-α were detected by quantitative real-time polymerase chain reaction ( a – c ). Protein expression of IL-1β, IL-8, and TNF-α was detected by enzyme-linked immunosorbent assay ( d – f ). Values are expressed as mean ± SEM of three independent experiments, each of which was run in triplicate. * P

    Article Snippet: MSU crystals can induce a variety of cytokines, including IL-1β, IL-6, IL-8, and TNF-α [ ].

    Techniques: Expressing, Concentration Assay, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

    Histological analysis of IL-1β staining in the placenta following MSU crystals treatment.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Uric acid crystals induces placental inflammation and alters trophoblast function via an IL-1-dependent pathway: implication for FGR

    doi: 10.4049/jimmunol.1601179

    Figure Lengend Snippet: Histological analysis of IL-1β staining in the placenta following MSU crystals treatment.

    Article Snippet: Both MSU crystals and IL-1β significantly induced cell death in cytotrophoblast cultures, as seen by the 3.3-fold and 2.7-fold increase, respectively, in the percentage of cells positive for the apoptotic marker M30 at 48h ( ).

    Techniques: Staining