Journal: Cellular and Molecular Immunology

Article Title: An animal model of NLRC4-associated autoinflammation and infantile enterocolitis reveals novel therapeutic strategies

doi: 10.1038/s41423-025-01355-x

Figure Lengend Snippet: NLRC4 V341A KI mice exhibit inflammasome hyperactivation. A Diagram illustrating the design and generation of NLRC4 V341A-Flag KI mice. B Mendelian inheritance analysis of NLRC4 KI offspring as indicated. C – G Six-day-old E2aCre-NLRC4 V341A fl/fl (KI) and E2aCre-NLRC4 +/+ (wild-type, WT) mice were used. C Western blot for detecting mouse NLRC4 and NLRC4-V341A-Flag expression in colon tissues of NLRC4 WT and KI mice to confirm the genotyping. D Western blot analysis of NLRC4 V341A-Flag expression in the indicated tissues probed with Flag and β-actin antibodies. SI small intestine; IECs intestinal epithelial cells; BMDMs bone marrow-derived macrophages. E Inflammasome activation in IECs from NLRC4 WT and KI mice was analyzed via western blotting with the indicated antibodies (FL GSDMD, full-length Gasdermin-D; GSDMD-N,N-terminal GSDMD. F IL-1β and IL-18 levels in colon explant cultures from NLRC4 WT and KI mice were measured via ELISA ( n = 6 per group). Colon tissues were cultured in 1 ml of medium overnight, and the supernatant was collected for analysis. G IL-1β and IL-18 levels in NLRC4 WT and KI mouse serum were measured via ELISA ( n = 10 per group). In ( F , G ), the data are shown as the mean ± SEM; ****, p < 0.0001 was determined by Student’s t test. The data are representative of at least three independent experiments

Article Snippet: The NLRC4 V341A-Flag flox/flox mice were generated by Cyagen Biosciences via Cyagen’s TurboKnockout ® gene targeting service through homologous recombination.

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

Journal: Cellular and Molecular Immunology

Article Title: An animal model of NLRC4-associated autoinflammation and infantile enterocolitis reveals novel therapeutic strategies

doi: 10.1038/s41423-025-01355-x

Figure Lengend Snippet: NLRC4 V341A KI mice develop autoinflammation. A Gross phenotype of NLRC4 WT (left) and NLRC4 KI (right) mice on day 6 after birth. B Body weight (left) and survival rate (right) of NLRC4 WT (NLRC4 +/+ ) and NLRC4 KI (NLRC4 KI/KI ) mice after birth were plotted ( n = 20 per group). P values were determined via two-way ANOVA and the Mantel‒Cox test. C Serum ferritin, hemoglobin and IL-6 levels in 6-day-old NLRC4 WT and NLRC4-KI mice were measured via ELISA ( n = 8 per group). D Chemical parameters of blood from NLRC4 WT and KI mice were measured as indicated. ALT, alanine aminotransferase; BUN, blood urea nitrogen; LDH, lactate dehydrogenase. Sera were pooled from 8–10 6-day-old mice per group. E Blood cells from 6-day-old NLRC4 WT and NLRC4-KI mice were counted for the indicated cellular parameters ( n = 5 per group). WBC, white blood cell; RBC, red blood cell. F Hemophagocytosis in the spleen was analyzed by flow cytometry. Splenocytes from 6-day-old NLRC4 WT and NLRC4 KI mice were first stained with fluorescence-conjugated CD11b (myeloid cells); then, the stained cells were incubated with unconjugated Ter119 antibodies to block corresponding surface antigens; and finally, the cells were permeabilized for intracellular staining of phagocytosed Ter119+ cells ( n = 3 per group). G Isolated macrophages from the spleens of 6-day-old NLRC4 WT and KI pups were analyzed for inflammatory gene expression as indicated by Q-PCR ( n = 4 per group). The data are shown as the means ± SEMs. p values were determined by Student’s t test ( C , E - G ), * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001. The data are representative of three independent experiments

Article Snippet: The NLRC4 V341A-Flag flox/flox mice were generated by Cyagen Biosciences via Cyagen’s TurboKnockout ® gene targeting service through homologous recombination.

Techniques: Enzyme-linked Immunosorbent Assay, Flow Cytometry, Staining, Fluorescence, Incubation, Blocking Assay, Isolation, Gene Expression

Journal: Cellular and Molecular Immunology

Article Title: An animal model of NLRC4-associated autoinflammation and infantile enterocolitis reveals novel therapeutic strategies

doi: 10.1038/s41423-025-01355-x

Figure Lengend Snippet: NLRC4 V341A KI mice develop infantile enterocolitis. A Representative H&E staining of small intestine and colon tissues from 6-day-old NLRC4 WT and KI mice as indicated. B Inflammatory cell infiltration in lamina propria from colon tissues of 6-day-old NLRC4 WT and KI mice was analyzed by flow cytometry ( n = 5 per group). C Colon tissues from 6-day-old NLRC4 WT and KI mice were analyzed for inflammatory gene expression as indicated by real-time PCR ( n = 4 per group). D IL-6 and TNF-α levels in the supernatants of colon explant cultures from 6-day-old NLRC4 WT and -KI mice were measured via ELISA ( n = 6 per group). E Representative images of gross colons from 6-day-old NLRC4 WT and KI mice. F Colon diameter, colon index (colon weight/body weight), wet/dry feces ratio and stool score of 6-day-old NLRC4 WT and KI mice are shown as indicated ( n = 10 per group). The data are shown as the means ± SEMs. p values were determined by Student’s t test, * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001. The data are representative of three independent experiments

Article Snippet: The NLRC4 V341A-Flag flox/flox mice were generated by Cyagen Biosciences via Cyagen’s TurboKnockout ® gene targeting service through homologous recombination.

Techniques: Staining, Flow Cytometry, Gene Expression, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: Cellular and Molecular Immunology

Article Title: An animal model of NLRC4-associated autoinflammation and infantile enterocolitis reveals novel therapeutic strategies

doi: 10.1038/s41423-025-01355-x

Figure Lengend Snippet: Gut barrier integrity is impaired in NLRC4 V341A KI mice. A Representative images of different histochemical stains as indicated; samples were colon (PAS staining) or otherwise distal small intestine tissues from 6-day-old NLRC4 WT and NLRC4 KI mice. Periodic acid–Schiff (PAS) staining was used for goblet cells, lysosomal staining was used for Paneth cells, TUNEL was used for cell death, and ZO-1 was used for tight junction assessment. B Western blot analysis of the type of cell death in distal small intestine tissues from 6-day-old NLRC4 WT and NLRC4 KI mice. Cleaved Gasdermin D (GSDMD-N) is indicative of pyroptosis, p-MLKL is a marker of necroptosis, and cleaved caspase 3 signifies apoptotic cell death. C Representative tight junction genes, as indicated, were analyzed via Q‒PCR. The samples were intestinal epithelial cells isolated from 6-day-old NLRC4 WT and NLRC4 KI mice ( n = 4 per group). p values were determined by Student’s t test, ** p < 0.01. The data are shown as the means ± SEMs. The data are representative of three independent experiments

Article Snippet: The NLRC4 V341A-Flag flox/flox mice were generated by Cyagen Biosciences via Cyagen’s TurboKnockout ® gene targeting service through homologous recombination.

Techniques: Staining, TUNEL Assay, Western Blot, Marker, Isolation

Journal: Cellular and Molecular Immunology

Article Title: An animal model of NLRC4-associated autoinflammation and infantile enterocolitis reveals novel therapeutic strategies

doi: 10.1038/s41423-025-01355-x

Figure Lengend Snippet: Kinetic analysis of AIFEC development in NLRC4 V341A KI pups. A Representative H&E staining of small intestine (left) and colon (right) tissues from 0-, 3-, and 6-day-old NLRC4 WT and NLRC4-KI mice. B IL-1β, IL-18 and IL-6 levels in the supernatants of colon explant cultures from NLRC4 WT and KI mice were measured via ELISA. C Colon tissues from 0-, 3-, and 6-day-old NLRC4 WT and NLRC4-KI mice were analyzed for inflammatory gene expression as indicated by Q‒PCR. D The levels of serum ferritin and IL-6 in 0-, 3-, and 6-day-old NLRC4 WT and NLRC4 KI mice were measured via ELISA. Sample size: n = 4/group. The data are shown as the means ± SEMs. p values were determined by Student’s t test, * p < 0.05, ** p < 0.01 and *** p < 0.001. The data are representative of three independent experiments

Article Snippet: The NLRC4 V341A-Flag flox/flox mice were generated by Cyagen Biosciences via Cyagen’s TurboKnockout ® gene targeting service through homologous recombination.

Techniques: Staining, Enzyme-linked Immunosorbent Assay, Gene Expression

Journal: Cellular and Molecular Immunology

Article Title: An animal model of NLRC4-associated autoinflammation and infantile enterocolitis reveals novel therapeutic strategies

doi: 10.1038/s41423-025-01355-x

Figure Lengend Snippet: IL18BP treatment improves survival in NLRC4 V341A KI pups with AIFEC. IL-18BP was administered to NLRC4 V341A KI pups via intraperitoneal (i.p.) injection at a dose of 8 mg/kg from day 1 to day 7 after birth. The NLRC4 WT (WT) and KI control groups received equivalent volumes of saline. Pups were monitored daily for weight and survival. A Body weights and survival rates of the indicated groups. B Representative H&E staining of colon tissues from 8-day-old and 22-day-old mice as indicated. C Stool scores of the indicated groups of mice on days 8 and 22 posttreatment. D Wet-to-dry feces ratios in the indicated groups. E Inflammatory gene expression in colon tissues from 8-day-old and 22-day-old mice was analyzed by real-time PCR. F Red blood cell (RBC) counts in blood samples from 8-day-old and 22-day-old mice. G Hemophagocytosis in the spleen was analyzed by flow cytometry, which revealed the percentage of myeloid cells that phagocytosed RBCs. H Serum levels of ferritin, IL-1β, and IL-18 in 8-day-old and 22-day-old mice were measured via ELISA. The data are presented as the means ± SEMs. Sample size: n = 5/group, except for panels E&G ( n = 4/group). Statistical significance was determined via Student’s t test (* p < 0.05, ** p < 0.01, *** p < 0.001). The results are representative of at least two independent experiments

Article Snippet: The NLRC4 V341A-Flag flox/flox mice were generated by Cyagen Biosciences via Cyagen’s TurboKnockout ® gene targeting service through homologous recombination.

Techniques: Injection, Control, Saline, Staining, Gene Expression, Real-time Polymerase Chain Reaction, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Cellular and Molecular Immunology

Article Title: An animal model of NLRC4-associated autoinflammation and infantile enterocolitis reveals novel therapeutic strategies

doi: 10.1038/s41423-025-01355-x

Figure Lengend Snippet: TNFα blockade dramatically alleviates AIFEC in NLRC4 V341A KI pups. Infliximab was administered to NLRC4 V341A KI pups via intraperitoneal (i.p.) injection at a dose of 5 mg/kg from day 1 to day 7 after birth. The control groups received equivalent volumes of isotype control antibodies. Pups were monitored daily for weight and survival. A Body weights and survival rates of the indicated groups. B Representative H&E staining of colon tissues from 8-day-old and 22-day-old mice as indicated. C Stool scores of the indicated groups of mice on days 8 and 22 posttreatment. D Wet-to-dry feces ratios in the indicated groups. E Inflammatory gene expression in colon tissues from 8-day-old and 22-day-old mice was analyzed by real-time PCR. F Red blood cell (RBC) counts in blood samples from 8-day-old and 22-day-old mice. G Hemophagocytosis in the spleen was analyzed by flow cytometry, which revealed the percentage of myeloid cells that phagocytosed RBCs. H Serum levels of ferritin, IL-1β, and IL-18 in 8-day-old and 22-day-old mice were measured via ELISA. The data are presented as the means ± SEMs. Sample size: n = 5/group, except for panels E&G ( n = 4/group). Statistical significance was determined via Student’s t test (* p < 0.05, ** p < 0.01, *** p < 0.001). The results are representative of at least two independent experiments

Article Snippet: The NLRC4 V341A-Flag flox/flox mice were generated by Cyagen Biosciences via Cyagen’s TurboKnockout ® gene targeting service through homologous recombination.

Techniques: Injection, Control, Staining, Gene Expression, Real-time Polymerase Chain Reaction, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Cellular and Molecular Immunology

Article Title: An animal model of NLRC4-associated autoinflammation and infantile enterocolitis reveals novel therapeutic strategies

doi: 10.1038/s41423-025-01355-x

Figure Lengend Snippet: Glucose supplementation protects NLRC4 V341A KI pups from AIFEC. Glucose was administered to NLRC4 V341A KI pups via intraperitoneal (i.p.) injection at a dose of 200 mg/kg from day 1 to day 7 after birth. The control groups received equivalent volumes of saline. Pups were monitored daily for weight and survival. A Body weights and survival rates of the indicated groups. B Representative H&E staining of colon tissues from 8-day-old and 22-day-old mice as indicated. C Stool scores of the indicated groups of mice on days 8 and 22 posttreatment. D Wet-to-dry feces ratios in the indicated groups. E Inflammatory gene expression in colon tissues from 8-day-old and 22-day-old mice was analyzed by real-time PCR. F Red blood cell (RBC) counts in blood samples from 8-day-old and 22-day-old mice. G Hemophagocytosis in the spleen was analyzed by flow cytometry, which revealed the percentage of myeloid cells that phagocytosed RBCs. H Serum levels of ferritin, IL-1β, and IL-18 in 8-day-old and 22-day-old mice were measured via ELISA. The data are presented as the means ± SEMs. Sample size: n = 5/group, except for ( E, G ) ( n = 4/group). Statistical significance was determined via Student’s t test (* p < 0.05, ** p < 0.01, *** p < 0.001). The results are representative of at least two independent experiments

Article Snippet: The NLRC4 V341A-Flag flox/flox mice were generated by Cyagen Biosciences via Cyagen’s TurboKnockout ® gene targeting service through homologous recombination.

Techniques: Injection, Control, Saline, Staining, Gene Expression, Real-time Polymerase Chain Reaction, Flow Cytometry, Enzyme-linked Immunosorbent Assay

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: bioRxiv

Article Title: TLR priming licenses NAIP inflammasome activation by immunoevasive ligands

doi: 10.1101/2023.05.04.539437

Figure Lengend Snippet: (A-B) WT, Nlrc4 -/- and Casp1/11 -/- murine BMDMs were infected with Δ yopJ Yp expressing either YopE 1-100 or YopE 1-100 fused to the C-terminal D0 portion of FliC from S. Tm or EPEC for four hours at an MOI of 20. (A) % cytotoxicity was measured via lactate dehydrogenase (LDH) release. (B) IL-1β release (pg/mL) was measured by ELISA. (C) BMDMs were infected with the indicated strains for two hours at an MOI of 20. Combined supernatants and cellular lysates were analyzed by immunoblotting for Casp1, GSDMD, and β−actin (loading control). Representative of three independent experiments. (D) List of mutants generated to assess contribution of C-terminal residues in S. Tm or EPEC FliC D0. Red indicates the base pair mutations compared to the WT sequence. (E, F) WT murine BMDMs were infected with the indicated strains for four hours at an MOI of 20. (E) % cytotoxicity was measured via LDH release. (F) IL-1β release (pg/mL) was measured by ELISA. (G) BMDMs were infected with the indicated strains for one hour at an MOI of 20. Combined supernatants and whole cell lysates were analyzed by immunoblotting for Casp1, GSDMD, and β−actin (loading control). Representative of three independent experiments. (A, E) Data shown are the pooled means ± SEM from three independent experiments. Paired t test was performed to assess statistical significance. (B, F) Data shown are the pooled means ± SEM from three independent experiments. Statistical significance was measured by performing an unpaired t test. ND=not detected; ns=not significant; *, P <0.05, **, P <0.01,***, P <0.001.

Article Snippet: To overexpress murine and human NLRC4 in immortalized Nlrc4 -/- murine myeloid progenitors, mscv2.2-IRESGFP retroviral vectors containing murine Nlrc4 (Addgene plasmid #60199) or human NLRC4 were used.

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

Journal: bioRxiv

Article Title: TLR priming licenses NAIP inflammasome activation by immunoevasive ligands

doi: 10.1101/2023.05.04.539437

Figure Lengend Snippet: (A and B) WT, Nlrc4 -/- , Casp1/11 -/- , and Naip5 -/- murine unprimed BMDMs or primed with 0.5 μg/mL Pam3CSK4 (Pam3) for 16 hours were infected with the indicated strains for four hours at an MOI of 20. (A) Cell death (% cytotoxicity) was measured via lactase dehydrogenase (LDH) release. (B) IL-1β release (pg/mL) was measured by ELISA. (C) BMDMs were infected with the indicated strains for two hours at an MOI of 20, TCA-precipitated supernatants and whole cell lysates were collected and combined. Samples were analyzed by immunoblot for Casp1, GSDMD, and β−actin (loading control). Image representative of three independent experiments. (D andE) WT murine unprimed BMDMs or primed with 0.5 μg/mL Pam3CSK4 for 16 hours were infected with the indicated strains for two hours at an MOI of 20. (D) % cytotoxicity was measured via LDH release. (E) IL-1β release (pg/mL) was measured by ELISA. (F) BMDMS were infected with the indicated strains for four hours at an MOI of 20. Combined supernatants and whole cell lysates were analyzed by immunoblotting for Casp1, GSDMD, and β−actin (loading control). Image representative of three independent experiments. (A and D) Data shown are pooled means ± SEM from three independent experiments. A paired t test was performed to assess statistical significance. (B, G) Data shown are representative of three independent experiments and are the combined means ± SEM. Statistical significance was measured by performing an unpaired t test. ns=not significant; *, P <0.05, **, P <0.01,***, P <0.001,****, P <0.001.

Article Snippet: To overexpress murine and human NLRC4 in immortalized Nlrc4 -/- murine myeloid progenitors, mscv2.2-IRESGFP retroviral vectors containing murine Nlrc4 (Addgene plasmid #60199) or human NLRC4 were used.

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

Journal: Biology of reproduction

Article Title: The alarmin S100A12 causes sterile inflammation of the human chorioamniotic membranes as well as preterm birth and neonatal mortality in mice†.

doi: 10.1093/biolre/ioab188

Figure Lengend Snippet: Figure 3. Incubation with S100A12 induces the transcriptional priming of inflammasomes in the human chorioamniotic membranes. The chorioamniotic membranes from women who delivered at term without labor were incubated with S100A12 (black dots) or PBS (control, white dots) for 24 h (n = 5–8 per group). Messenger RNA abundance of NLRP1 (A), NLRP3 (B), NLRC4 (C), AIM2 (D), NOD1 (E), and NOD2 (F). Relative gene expression is presented as –CT values. The P-values were determined by paired student’s t-tests.

Article Snippet: Enzyme-linked immunosorbent assays The concentrations of IL-6, IL-8, and mature IL-1β in tissue culture supernatants (n = 12–15 per group), of NLRP1, NLRP3, AIM2, NLRC4, NOD1, and NOD2 in tissue extracts (n = 10–14 per group), and of caspase-1 in conditioned media-treated tissue extracts (n = 6 per group) were measured, using specific and sensitive immunoassays (IL-6, IL-8, and IL-1β: enzyme-linked immunosorbent assay (ELISA) kits from R&D Systems, Minneapolis, MN, USA; NLRP1, NLRP3, NLRC4, NOD1, and NOD2: ELISA kits from Cusabio, Wuhan, Hubei, P.R.

Techniques: Incubation, Control, Gene Expression

Journal: Biology of reproduction

Article Title: The alarmin S100A12 causes sterile inflammation of the human chorioamniotic membranes as well as preterm birth and neonatal mortality in mice†.

doi: 10.1093/biolre/ioab188

Figure Lengend Snippet: Figure 4. Incubation with S100A12 increases the protein expression of inflammasome sensor molecules in the human chorioamniotic membranes. The chorioamniotic membranes from women who delivered at term without labor were incubated with S100A12 (black dots) or PBS (control, white dots) for 24 h (n = 10–14 per group). The concentrations of NLRP1 (A), NLRP3 (B), NLRC4 (C), AIM2 (D), NOD1 (C), and NOD2 (F) in the tissue lysates of human chorioamniotic membrane samples were evaluated by specific ELISA kits. The P-values were determined by Wilcoxon signed-rank tests.

Article Snippet: Enzyme-linked immunosorbent assays The concentrations of IL-6, IL-8, and mature IL-1β in tissue culture supernatants (n = 12–15 per group), of NLRP1, NLRP3, AIM2, NLRC4, NOD1, and NOD2 in tissue extracts (n = 10–14 per group), and of caspase-1 in conditioned media-treated tissue extracts (n = 6 per group) were measured, using specific and sensitive immunoassays (IL-6, IL-8, and IL-1β: enzyme-linked immunosorbent assay (ELISA) kits from R&D Systems, Minneapolis, MN, USA; NLRP1, NLRP3, NLRC4, NOD1, and NOD2: ELISA kits from Cusabio, Wuhan, Hubei, P.R.

Techniques: Incubation, Expressing, Control, Membrane, Enzyme-linked Immunosorbent Assay