Journal: Cancer biology & therapy

Article Title: Curcumin activates NLRC4, AIM2, and IFI16 inflammasomes and induces pyroptosis by up-regulated ISG3 transcript factor in acute myeloid leukemia cell lines.

doi: 10.1080/15384047.2022.2058862

Figure Lengend Snippet: Figure 1. Curcumin induces the expression of AIM2, IFI16, and NLRC4 inflammasomes in leukemia cells U937. (a) Cene expression chip analyzed the expression of NLRP1, NLRP3, NLRC4, AIM2, IFI16, ASC, and GSDMD in U937 treated with curcumin (10 μmol/L) for 24 h; (b) validation of NLRC4, AIM2, and IFI16 expression in U937 when treated with curcumin for 24 h by RT-PCR. Each value represents the mean ± SD (*P < .05, **P < .01, ***P < .001); (c) western blot array analyzed protein level of NLRC4, AIM2, and IFI16 h in U937 after treated with curcumin for 24 h.

Article Snippet: The antibodies against AIM2, NLRC4, and IFI16 were purchased from ABclonal Technology (Wuhan, China) Co.,Ltd.

Techniques: Expressing, Biomarker Discovery, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Human & experimental toxicology

Article Title: Effects of lysophosphatidic acid receptor 5 on NLRC4 inflammasome in brain tissues of transient cerebral ischemia/reperfusion rat.

doi: 10.1177/09603271221078870

Figure Lengend Snippet: Figure 1. Graphical abstract of this design. There were two experimental designs, named as Experiment 1 (A) and Experiment 2 (B). Firstly, tMACO rat was performed at ischemia of 2h and reperfusion for different time points, and we selected the optimal time point of reperfusion for the following study according to the peak value of NLRC4 at 4h. Secondly, LPA5 siRNA and its negative control vectors were icv. injected after reperfusion of 4h, and after 24h, the brains were collected for the further experiments.

Article Snippet: Rabbit anti-rat monoclonal antibody: LPA5/GPR92 (1:1000, orb157371, Biorbyt), NLRC4 (1:1000, orb412596, Biorbyt), AIM2 (1: 800, orb330613, Biorbyt), caspase-1 (1:900, orb213641, Biorbyt), cleaved caspase-1 p20 (1:1000, WL02996a, Wanleibio), mature IL-1β (1:500, WL00891, Wanleibio), precursor IL-1β (1:1000, obr500658, Biorbyt), and β-actin (1:2000, ab8227, Abcam).

Techniques: Negative Control, Injection

Journal: Human & experimental toxicology

Article Title: Effects of lysophosphatidic acid receptor 5 on NLRC4 inflammasome in brain tissues of transient cerebral ischemia/reperfusion rat.

doi: 10.1177/09603271221078870

Figure Lengend Snippet: Figure 2. After different reperfusion (4, 8, 12, and 24h), immunofluorescence experiment was implemented to detect expression of LPA5 and NLRC4 in brain tissues of tMCAO model. All experiments were repeated at least 4 times. The scale bar is 20 μm. Compared with sham group, *p < 0.05, **p < 0.01; compared with CI/R-4h group, &&p < 0.01; compared with CI/R-8h,^p < 0.05,^^p < 0.01; compared with CI/R-12, %p < 0.05.

Article Snippet: Rabbit anti-rat monoclonal antibody: LPA5/GPR92 (1:1000, orb157371, Biorbyt), NLRC4 (1:1000, orb412596, Biorbyt), AIM2 (1: 800, orb330613, Biorbyt), caspase-1 (1:900, orb213641, Biorbyt), cleaved caspase-1 p20 (1:1000, WL02996a, Wanleibio), mature IL-1β (1:500, WL00891, Wanleibio), precursor IL-1β (1:1000, obr500658, Biorbyt), and β-actin (1:2000, ab8227, Abcam).

Techniques: Expressing

Journal: Human & experimental toxicology

Article Title: Effects of lysophosphatidic acid receptor 5 on NLRC4 inflammasome in brain tissues of transient cerebral ischemia/reperfusion rat.

doi: 10.1177/09603271221078870

Figure Lengend Snippet: Figure 3. Western blot was used to detect the expression of LPA5, NLRC4, AIM2, Cleaved caspase-1, and Mature IL-1β protein. Image J 1.49p software was used to analyze the grey values of each protein band. All experiments were repeated at least 4 times. Compared with Sham group, **p < 0.01; compared with CI/R-4h group, &p < 0.05, &&p < 0.01; compared with CI/R-8h,^p < 0.05,^^p < 0.01; compared with CI/R-12, %%p < 0.01.

Article Snippet: Rabbit anti-rat monoclonal antibody: LPA5/GPR92 (1:1000, orb157371, Biorbyt), NLRC4 (1:1000, orb412596, Biorbyt), AIM2 (1: 800, orb330613, Biorbyt), caspase-1 (1:900, orb213641, Biorbyt), cleaved caspase-1 p20 (1:1000, WL02996a, Wanleibio), mature IL-1β (1:500, WL00891, Wanleibio), precursor IL-1β (1:1000, obr500658, Biorbyt), and β-actin (1:2000, ab8227, Abcam).

Techniques: Western Blot, Expressing, Software

Journal: Human & experimental toxicology

Article Title: Effects of lysophosphatidic acid receptor 5 on NLRC4 inflammasome in brain tissues of transient cerebral ischemia/reperfusion rat.

doi: 10.1177/09603271221078870

Figure Lengend Snippet: Figure 5. Immunofluorescence assay was performed to detect the LAP5 and NLRC4 expression in brain tissues of CI/R rats after silencing LPA5. All experiments were repeated at least 4 times. The scale bar is 20 μm. Compared with Sham group, *p < 0.05, **p < 0.01; compared with CI/R-4h group, &&p < 0.01; compared with si-control, %%p < 0.01.

Article Snippet: Rabbit anti-rat monoclonal antibody: LPA5/GPR92 (1:1000, orb157371, Biorbyt), NLRC4 (1:1000, orb412596, Biorbyt), AIM2 (1: 800, orb330613, Biorbyt), caspase-1 (1:900, orb213641, Biorbyt), cleaved caspase-1 p20 (1:1000, WL02996a, Wanleibio), mature IL-1β (1:500, WL00891, Wanleibio), precursor IL-1β (1:1000, obr500658, Biorbyt), and β-actin (1:2000, ab8227, Abcam).

Techniques: Expressing, Control

Journal: Human & experimental toxicology

Article Title: Effects of lysophosphatidic acid receptor 5 on NLRC4 inflammasome in brain tissues of transient cerebral ischemia/reperfusion rat.

doi: 10.1177/09603271221078870

Figure Lengend Snippet: Figure 6. Western blot assay was carried out to determine LPA5, NLRC4, AIM2, Cleaved caspase-1, and Mature IL-1β protein after LPA5 siRNA treatment (n = 4). (A) Western blot was employed to detect LPA5 and NLRC4 expression. (B) Western blot was used to analyze AIM2, Cleaved caspase-1, and Mature IL-1β protein. Compared with Sham group, *p < 0.05, **p < 0.01; compared with CI/R-4h group, &&p < 0.01; compared with si-control, %%p < 0.01.

Article Snippet: Rabbit anti-rat monoclonal antibody: LPA5/GPR92 (1:1000, orb157371, Biorbyt), NLRC4 (1:1000, orb412596, Biorbyt), AIM2 (1: 800, orb330613, Biorbyt), caspase-1 (1:900, orb213641, Biorbyt), cleaved caspase-1 p20 (1:1000, WL02996a, Wanleibio), mature IL-1β (1:500, WL00891, Wanleibio), precursor IL-1β (1:1000, obr500658, Biorbyt), and β-actin (1:2000, ab8227, Abcam).

Techniques: Western Blot, Expressing, Control

Journal: The Journal of Experimental Medicine

Article Title: LRRK2 promotes the activation of NLRC4 inflammasome during Salmonella Typhimurium infection

doi: 10.1084/jem.20170014

Figure Lengend Snippet: LRRK2 is critical for NLRC4 inflammasome activation. (a and b) LPS-primed WT and Lrrk2 −/− peritoneal macrophages were treated with 1 µg/ml LFn-PrgJ and anthrax-protective antigen (PA; a) or 1 µg/ml LFn-flagellin + anthrax-protective antigen for 1 h (b). Cell lysates and culture supernatants (Sup) were collected and immunoblotted with the indicated antibodies. (c) Peritoneal macrophages from littermate control (WT) and Lrrk2 −/− mice were infected with S. Typhimurium at an MOI of 100 for 2 h. Cell lysates and culture supernatants were collected and immunoblotted with the indicated antibodies. (d) ELISA of TNF-α in cell-free supernatants from WT and Lrrk2 −/− peritoneal macrophages that were either infected with S. Typhimurium at an MOI of 100 for 2 h or pretreated with LPS (500 ng/ml) for 4 h followed by stimulation with LFn-flagellin + anthrax-protective antigen (1 µg/ml), LFn-PrgJ anthrax-protective antigen (1 µg/ml), ATP (5 mM), or nigericin (20 µM) for 1 h. (e) WT and Lrrk2 −/− peritoneal macrophages were primed with 500 ng/ml LPS for 4 h and then treated with ATP (5 mM) or nigericin (20 µM) for 1 h. Cell lysates and culture supernatants were collected and immunoblotted with the indicated antibodies. “Ctrl” indicates the control group. (f) ELISA of IL-1β in cell-free supernatants from WT and Lrrk2 −/− peritoneal macrophages that were treated as stated in d. Data are shown as means ± SEM; *, P < 0.05; **, P < 0.01; ***, P < 0.001; determined by Student’s t test; all conditions were determined in triplicate. In each panel, data are representative of at least three independent experiments.

Article Snippet: Anti-NLRC4 and NLRC4 (p-Ser533) antibodies were from ECM Biosciences.

Techniques: Activation Assay, Control, Infection, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Experimental Medicine

Article Title: LRRK2 promotes the activation of NLRC4 inflammasome during Salmonella Typhimurium infection

doi: 10.1084/jem.20170014

Figure Lengend Snippet: LRRK2 promotes ASC speck formation and assembly of NLRC4 inflammasome. (a) Peritoneal macrophages from littermate control (WT) and Lrrk2 −/− mice were infected with S . Typhimurium at an MOI of 50 for 1 or 2 h. After cross-linking with DTBP, cell lysates were immunoprecipitated (IP) with anti-ASC antibody and immunoblotted with the indicated antibodies. Whole-cell lysates are shown as the input. (b and c) LPS-primed WT and Lrrk2 −/− peritoneal macrophages were either infected with S. Typhimurium at an MOI of 50 (b) or cytosolic delivery of 1 µg/ml LFn-flagellin (c). ASC speck formation was assayed by ASC immunofluorescent staining, and cells were counterstained by DAPI (blue). Fluorescent images were analyzed by confocal microscopy. Percentages of macrophages containing ASC foci was quantified (right), with at least 200 cells counted in each experiment. Each condition was performed in triplicate. Quantitative data are shown as means ± SEM; ***, P < 0.001; determined by Student’s test. Arrowheads mark ASC specks. Bars, 10 µm. (d) Peritoneal macrophages from littermate control (WT) and Lrrk2 −/− mice were infected with S. Typhimurium at an MOI of 50 for 1 h. Cells were dissolved with Triton X-100–containing buffer followed by cross-linkage of insoluble fractions with DSS to capture ASC oligomers. Immunoblots of those insoluble fractions (Insoluble + DSS) and soluble fractions were detected with an antibody to ASC. (e) Immunoblot analysis of ASC in cross-linked Triton X-100–insoluble fractions from LPS-primed WT and Lrrk2 −/− macrophages treated with 1 µg/ml LFn-flagellin + anthrax-protective antigen (PA) for 30 min. All results are representative of three independent experiments.

Article Snippet: Anti-NLRC4 and NLRC4 (p-Ser533) antibodies were from ECM Biosciences.

Techniques: Control, Infection, Immunoprecipitation, Staining, Confocal Microscopy, Western Blot

Journal: The Journal of Experimental Medicine

Article Title: LRRK2 promotes the activation of NLRC4 inflammasome during Salmonella Typhimurium infection

doi: 10.1084/jem.20170014

Figure Lengend Snippet: LRRK2 directly interacts with NLRC4 to promote inflammasome assembly. (a) Peritoneal macrophages from WT and Lrrk2 −/− mice were infected with S. Typhimurium at an MOI of 50 for 45 min. Uninfected cells were included as controls. Cell lysates were then immunoprecipitated (IP) using anti-LRRK2 antibody and were immunoblotted with the indicated antibodies. Whole-cell lysates are shown as the input. (b) LRRK2 interacts with NLRC4 but not NLRP3 and ASC. The HEK293T cells were transfected with Myc-tagged LRRK2 and cotransfected with/without Flag-tagged NLRC4, NLRP3, or ASC as indicated. Cell lysates were immunoprecipitated with anti-Flag and analyzed by immunoblot. (c) In vitro pull-down assay using recombinant LRRK2 and NLRC4 proteins: 50 ng purified 3×Flag-NLRC4 protein was incubated with 200 ng recombinant LRRK2 protein followed by immunoprecipitation with anti-LRRK2 antibody and Western blot analysis. (d) Flag-tagged WT full-length LRRK2 or different LRRK2 domains (LRR, ROC, C terminus of ROC [COR], kinase, and WD40) were coexpressed with Myc-tagged NLRC4 in 293T cells, immunoprecipitated with anti-Flag, and analyzed by immunoblot. (e) Flag-tagged full-length NLRC4 and the NLRC4 mutants ΔLRR (NLRC4 lacking LRR domain), ΔCARD (NLRC4 lacking CARD), or only nucleotide-binding domain (NBD) domain were coexpressed with Myc-tagged LRRK2 in HEK293T cells, immunoprecipitated with anti-Flag, and analyzed by immunoblot. (f) Flag-tagged NLRC4 LRR domain was coexpressed with Myc-tagged LRRK2 WD40 domain in 293T cells followed by immunoprecipitation with anti-Flag antibody and immunoblot analysis. (g) LPS-primed peritoneal macrophages from littermate control (WT) and Lrrk2 −/− mice were treated with LRRK2 inhibitor GSK2578215A (GSK) at 2 µM for 1 h and then infected with S. Typhimurium at an MOI of 50 for 1 h. Cell lysates were subjected to co-immunoprecipitation with anti-LRRK2 antibody followed by Western blotting. Whole-cell lysates are shown as the input. All the results are representative of at least three independent experiments.

Article Snippet: Anti-NLRC4 and NLRC4 (p-Ser533) antibodies were from ECM Biosciences.

Techniques: Infection, Immunoprecipitation, Transfection, Western Blot, In Vitro, Pull Down Assay, Recombinant, Purification, Incubation, Binding Assay, Control

Journal: The Journal of Experimental Medicine

Article Title: LRRK2 promotes the activation of NLRC4 inflammasome during Salmonella Typhimurium infection

doi: 10.1084/jem.20170014

Figure Lengend Snippet: LRRK2 kinase activity is required for NLRC4 inflammasome activation. (a) LPS-primed peritoneal macrophages were treated with LRRK2 inhibitors 2 µM LRRK2–IN-1 (IN-1) or 2 µM GSK2578215A for 1 h and then infected with S. Typhimurium at an MOI of 50 for 1 h. Cells were lysed with Triton X-100–containing buffer followed by cross-linkage of insoluble fractions with DSS to capture ASC oligomers. Insoluble fractions (Insoluble + DSS) and soluble fractions were detected with antibody to ASC by immunoblot. (b and c) LPS-primed macrophages were treated with LRRK2 inhibitors either by 2 µM LRRK2–IN-1 or 2 µM GSK2578215A for 1 h and then infected with S. Typhimurium at an MOI of 100 for 2 h (b) or stimulated with 1 µg/ml LFn-flagellin and anthrax-protective antigen (PA) for 1 h (c). Cell lysates and culture supernatants (Sup) were collected and blotted with the indicated antibodies. (d and e) Reconstitution of the NLRC4 inflammasome activation in HEK293T cells: HEK293T cells were plated in six-well microplates. The cells were transfected with plasmids expressing Flag-tagged human pro–IL-1β (500 ng/well), pro–caspase-1 (300 ng/well), and NLRC4 (500 ng/well) with or without plasmid encoding Myc-tagged LRRK2 (1,500 ng/well) or its G2019S or D2017A mutants for 24 h. IL-1β cleavage was assessed by immunoblot (d) and ELISA analysis of IL-1 β level (e) in the supernatants. Data are shown as means ± SEM; ***, P < 0.001; determined by one-way ANOVA followed by Bonferroni’s post hoc test. (f) LRRK2 -deficient iBMDMs were generated by CRISPR-Cas9–mediated deletion. For rescue experiments, Myc-tagged human LRRK2 WT, G2019S, or D2017A mutants were stably expressed in the LRRK2 -deficient iBMDMs. These cells were primed with LPS and then infected with S. Typhimurium at an MOI of 50 for 1 h. Cells were dissolved with Triton X-100–containing buffer followed by cross-linkage of insoluble fractions with DSS to capture ASC oligomers. Insoluble fractions (Insoluble + DSS) and soluble fractions were detected with antibody to ASC by immunoblot. (g) LRRK2 -deficient iBMDMs were restored with Myc-tagged human LRRK2 WT, G2019S, or D2017A mutants and then infected with S. Typhimurium at an MOI of 100 for 2 h. Cell lysates and culture supernatants were collected and blotted with the indicated antibodies. All the data are representative of three independent experiments.

Article Snippet: Anti-NLRC4 and NLRC4 (p-Ser533) antibodies were from ECM Biosciences.

Techniques: Activity Assay, Activation Assay, Infection, Western Blot, Transfection, Expressing, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Generated, CRISPR, Stable Transfection

Journal: The Journal of Experimental Medicine

Article Title: LRRK2 promotes the activation of NLRC4 inflammasome during Salmonella Typhimurium infection

doi: 10.1084/jem.20170014

Figure Lengend Snippet: LRRK2 phosphorylates NLRC4 Ser533 for NLRC4 inflammasome activation. (a–c) BMDMs from littermate control (WT) and Lrrk2 −/− mice were infected with S. Typhimurium at an MOI of 50 for 1 h (a) and then stimulated with 1 µg/ml LFn-flagellin and anthrax-protective antigen (PA) for 30 min (b) or 1 µg/ml LFn-PrgJ and PA (c) for 20 min. Cell lysates were collected and blotted with the indicated antibodies. Relative quantitation of NLRC4 phosphorylation was performed by ImageJ. Each relative pNLRC4 value was normalized to total NLRC4 protein and to the WT control. Each condition was performed at least in triplicate. Data are shown as means ± SEM; *, P < 0.05; determined by Student’s t test. (d) Flag-tagged WT or NLRC4 mutant S533A were coexpressed with Myc-tagged LRRK2 in HEK293T cells. Cell lysates were collected and blotted with the indicated antibodies. (e) In vitro kinase assay. Immunoprecipitated Myc-tagged LRRK2 WT, G2019S, or D2017A were mixed with purified Flag-tagged WT or NLRC4 mutant S533A (50 ng as substrates), respectively. pNLRC4 and protein inputs were analyzed by immunoblot. (f) In vitro kinase assay. Purified human LRRK2 (200 ng) or PKCδ protein (50 ng) were mixed with immunoprecipitated Flag-tagged WT or NLRC4 mutant S533A (50 ng as substrates), respectively. NLRC4 phosphorylation was analyzed by immunoblot. (g) In vitro kinase assay with inhibitors. Purified recombinant LRRK2 (200 ng) or PKCδ protein (50 ng) were treated with LRRK2 inhibitor GSK2578215A (GSK) and mixed with purified Flag-tagged WT NLRC4 (substrates). NLRC4 phosphorylation and protein inputs were analyzed by immunoblot. (h) Primary BMDMs from littermate control (WT) and Lrrk2 −/− mice were transfected with scramble control siRNA (siCon) or siRNA-targeting PKCδ (siPKCδ) and stimulated with LPS plus S. Typhimurium infection. Cell lysates and culture supernatants (Sup) were collected and blotted with the indicated antibodies. All data are representative of three independent experiments.

Article Snippet: Anti-NLRC4 and NLRC4 (p-Ser533) antibodies were from ECM Biosciences.

Techniques: Activation Assay, Control, Infection, Quantitation Assay, Phospho-proteomics, Mutagenesis, In Vitro, Kinase Assay, Immunoprecipitation, Purification, Western Blot, Recombinant, Transfection

Journal: Nutrition & metabolism

Article Title: Docosahexaenoic acid ameliorates palmitate-induced lipid accumulation and inflammation through repressing NLRC4 inflammasome activation in HepG2 cells.

doi: 10.1186/1743-7075-9-34

Figure Lengend Snippet: Figure 3 DHA inhibits PA-induced NLRC4 mRNA expression. Cells were treated accordingly and then NLRC4 mRNA was measured by real-time PCR The data are normalized using β-actin as control and expressed as fold over untreated cells. The experiment was repeated 4 times. *P < 0.05, **P < 0.01 compared with untreated cells; #P < 0.01 versus 0.4 mM PA-treated cells.

Article Snippet: The membranes were blocked with 5% BSA in TBS for 1 h at room temperature and incubated overnight at 4°C using the following primary antibodies: 1:1000 rabbit anti β-actin, 1:500 NLRC4 (Santa Cruz, CA, USA), 1:1000 caspase-1 and 1:500 IL-1β, followed by 1:2000 dilution of goat anti-rabbit horseradish peroxidaselabeled antibody.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Control

Journal: Nutrition & metabolism

Article Title: Docosahexaenoic acid ameliorates palmitate-induced lipid accumulation and inflammation through repressing NLRC4 inflammasome activation in HepG2 cells.

doi: 10.1186/1743-7075-9-34

Figure Lengend Snippet: Figure 4 DHA reverses PA-induced lipid accumulation through repressing NLRC4 expression. (A) Generation of a HepG2 cell model with specific NLRC4 knockdown (NLRC4+/−). NLRC4 and β-actin protein levels were evaluated by immunoblotting. (B) Representative Oil Red O staining of wild-type (WT) and NLRC4+/−HepG2 cells were treated with different dose of PA with or without DHA preincubation wild-type (WT) and NLRC4+/−cells are shown. Cells were examined by light microscopy at a magnification of 400×; (C) Intracellular TG content was measured by an ELISA assay. TG concentration was normalized by protein content. Data are shown as the mean ± S.E.; n = 4. *P < 0.05, **P < 0.01 compared with untreated cells; #P < 0.01 versus 0.4 mM PA-treated cells.

Article Snippet: The membranes were blocked with 5% BSA in TBS for 1 h at room temperature and incubated overnight at 4°C using the following primary antibodies: 1:1000 rabbit anti β-actin, 1:500 NLRC4 (Santa Cruz, CA, USA), 1:1000 caspase-1 and 1:500 IL-1β, followed by 1:2000 dilution of goat anti-rabbit horseradish peroxidaselabeled antibody.

Techniques: Expressing, Knockdown, Western Blot, Staining, Light Microscopy, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: Nutrition & metabolism

Article Title: Docosahexaenoic acid ameliorates palmitate-induced lipid accumulation and inflammation through repressing NLRC4 inflammasome activation in HepG2 cells.

doi: 10.1186/1743-7075-9-34

Figure Lengend Snippet: Figure 5 DHA reverses PA-induced inflammation through repressing NLRC4 expression. WT and NLRC4+/−HepG2 cells were treated with different dose of PA with or without DHA preincubation and then cell-free culture supernatants were assayed for IL-1β (A), IL-18 (B), TNF-α (C) and MCP-1 (D) by commercial ELISA kits. The data represent a mean of 4 experiments. *P < 0.05, **P < 0.01 compared with untreated cells; #P < 0.01 versus 0.4 mM PA-treated cells.

Article Snippet: The membranes were blocked with 5% BSA in TBS for 1 h at room temperature and incubated overnight at 4°C using the following primary antibodies: 1:1000 rabbit anti β-actin, 1:500 NLRC4 (Santa Cruz, CA, USA), 1:1000 caspase-1 and 1:500 IL-1β, followed by 1:2000 dilution of goat anti-rabbit horseradish peroxidaselabeled antibody.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay

Journal: Nutrition & metabolism

Article Title: Docosahexaenoic acid ameliorates palmitate-induced lipid accumulation and inflammation through repressing NLRC4 inflammasome activation in HepG2 cells.

doi: 10.1186/1743-7075-9-34

Figure Lengend Snippet: Figure 6 DHA inhibits caspase-1 activity and IL-1β maturation. WT and NLRC4+/−HepG2 cells were treated with different dose of PA with or without DHA preincubation and cells were homogenized in lysis buffer. (A) Caspase-1 activity was determined using colorimetric assay based on the cleavage of substrate Ac-YVAD-pNA. The results were expressed as fold over untreated WT cells. *P < 0.05, **P < 0.01 compared with untreated cells; #P < 0.01 versus 0.4 mM PA-treated cells. (B) Immunoblot analysis showing caspase-1 cleavage and active IL-1β accumulation. This gel is the representative image of three independent experiments (upper panel). Quantified data of protein levels indicates in lower panel. The values of density of proteins were all justified with β-actin. The relative density ratios of untreated WT cells were set at a value of 1.0. The values represent the mean ± S.E (*P < 0.05, **P < 0.01 compared with WT cells treated with 0.4 mm PA for the ratio of caspase-1/procaspase-1; #P < 0.01 versus 0.4 mM PA-treated WT cells for the ratio of IL-1β/pro-IL-1β).

Article Snippet: The membranes were blocked with 5% BSA in TBS for 1 h at room temperature and incubated overnight at 4°C using the following primary antibodies: 1:1000 rabbit anti β-actin, 1:500 NLRC4 (Santa Cruz, CA, USA), 1:1000 caspase-1 and 1:500 IL-1β, followed by 1:2000 dilution of goat anti-rabbit horseradish peroxidaselabeled antibody.

Techniques: Activity Assay, Lysis, Colorimetric Assay, Western Blot