nf κb pathway inhibitor bay 11 7082  (MedChemExpress)

Journal: Advanced Biology

Article Title: HMGB1 Derived from the Pyroptotic Microenvironment Promotes Macrophage Extracellular Traps in Hirschsprung‐Associated Enterocolitis

doi: 10.1002/adbi.202400761

Figure Lengend Snippet: The TLR4‐P38 MAPK/P65 NF‐kB signaling pathways mediate the‐pyroptotic‐microenvironment‐induced MET formation. A) Western blot analysis of p‐ERK, p‐p38, p‐JNK and p‐p65 levels in macrophages cocultured with pyroptotic‐CM for 4 h. B,C) Macrophages were pretreated with inhibitors of the ERK, JNK, p38 MAPK, and p65 NF‐kB pathways prior to incubation with pyroptotic‐CM. The MET formation by macrophages was evaluated with SYTOX Green staining and detected by flow cytometry. The representative images are shown in B and the quantification of B is shown in C (one‐way ANOVA followed by Tukey's multiple‐comparison test, n = 3 per group). D) Macrophages were pretreated with inhibitors of TLR2, TLR4, TLR9 and RAGE prior to coculture with pyroptotic‐CM, and the p‐p38 and p‐p65 levels in macrophages were measured by western blotting. E,F) The MET formation of macrophages pretreated with inhibitors of TLR2, TLR4 and RAGE prior to incubation with pyroptotic‐CM was evaluated with SYTOX Green staining and detected by flow cytometry. The representative images are shown in E and the quantification of E is shown in F (one‐way ANOVA followed by Tukey's multiple‐comparison test, n = 3 per group). Data are expressed as mean ± SD. ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The inhibitors used in this study included an HMGB1 antagonist (HY‐N0184, MCE, USA), a p38 MAPK pathway inhibitor (HY‐12839, MCE, USA), a p65 NF‐kB pathway inhibitor (HY‐138537, MCE, USA), an ERK pathway inhibitor (HY‐112287, MCE, USA), a JNK inhibitor (HY‐12041, MCE, USA), a TLR2 antagonist (HY‐112146, MCE, USA), a TLR4 antagonist (HY‐11109, MCE, USA), a TLR9 antagonist ( HY131952 , MCE, USA), and a RAGE antagonist (HY‐P2268).

Techniques: Protein-Protein interactions, Western Blot, Incubation, Staining, Flow Cytometry, Comparison

Journal: Advanced Biology

Article Title: HMGB1 Derived from the Pyroptotic Microenvironment Promotes Macrophage Extracellular Traps in Hirschsprung‐Associated Enterocolitis

doi: 10.1002/adbi.202400761

Figure Lengend Snippet: HMGB1 induces MET formation through TLR4‐P38 MAPK/P65 NF‐kB signaling pathways in macrophages. A‐C) BMDMs isolated from mice were stimulated with HMGB1 or PBS. Then, differentially expressed genes (DEGs) were analyzed by RNA sequencing. (A) The number of DEGs in the HMGB1 group vs. the PBS group. Red represented upregulated DEGs, and blue downregulated DEGs. (B) KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analyses of the upregulated DEGs. The dot size represents the number of DEGs, and the dot color represents the corresponding p value. (C) Scatter plot showing DEGs in the HMGB1 group vs. the PBS group. Genes were plotted based on their expression levels. Red and green dots represented up and downregulated genes, respectively. D‐I) qRT‐PCR analysis of the indicated genes in macrophages treated with HMGB1 or PBS (Unpaired t‐test, n = 3 per group). J) Western blot analysis of p‐ERK, p‐p38, p‐JNK and p‐p65 levels in macrophages cocultured with HMGB1. K) Macrophages were pretreated with inhibitors of TLR4 prior to incubation with HMGB1, and the p‐p38 and p‐p65 levels in macrophages was measured by western blotting. Data are expressed as mean ± SD. * p < 0.05, ** p < 0.01, **** p < 0.0001.

Article Snippet: The inhibitors used in this study included an HMGB1 antagonist (HY‐N0184, MCE, USA), a p38 MAPK pathway inhibitor (HY‐12839, MCE, USA), a p65 NF‐kB pathway inhibitor (HY‐138537, MCE, USA), an ERK pathway inhibitor (HY‐112287, MCE, USA), a JNK inhibitor (HY‐12041, MCE, USA), a TLR2 antagonist (HY‐112146, MCE, USA), a TLR4 antagonist (HY‐11109, MCE, USA), a TLR9 antagonist ( HY131952 , MCE, USA), and a RAGE antagonist (HY‐P2268).

Techniques: Protein-Protein interactions, Isolation, RNA Sequencing, Expressing, Quantitative RT-PCR, Western Blot, Incubation

Journal: Advanced Biology

Article Title: HMGB1 Derived from the Pyroptotic Microenvironment Promotes Macrophage Extracellular Traps in Hirschsprung‐Associated Enterocolitis

doi: 10.1002/adbi.202400761

Figure Lengend Snippet: MET formation enhances inflammatory responses and induces damage to CECs. A,B) BMDMs were treated with pyroptotic‐CM to induce MET formation, followed by co‐incubating with WT untreated BMDM. TNF‐α and IL‐1β mRNA levels in the WT BMDM were then measured by qRT‐PCR (Unpaired t‐test, n = 3 per group). C) CT26 cells were treated with METs or PBS for 0, 12, 24, 48 and 72 h, after which cell viability was detected by CCK‐8 assays (two‐way ANOVA followed by Sidak's multiple‐comparison test, n = 6 per group). D) CT26 cells were stimulated with METs or PBS and the ROS production was measured with DCFH‐DA staining and detected by microplate reader (Unpaired t‐test, n = 4 per group). E,F) Levels of ROS in CT26 cells after treatment of METs or PBS were assessed by DCFH‐DA staining and detected by flow cytometry. The images of flow cytometry are shown in E and the quantification of E is shown in F (Unpaired t‐test, n = 3 per group). G) Immunoblot analysis of p‐p65, GSDMD, and caspase‐1 protein in lysates of CT26 cells treated with METs. Data are expressed as mean ± SD. ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The inhibitors used in this study included an HMGB1 antagonist (HY‐N0184, MCE, USA), a p38 MAPK pathway inhibitor (HY‐12839, MCE, USA), a p65 NF‐kB pathway inhibitor (HY‐138537, MCE, USA), an ERK pathway inhibitor (HY‐112287, MCE, USA), a JNK inhibitor (HY‐12041, MCE, USA), a TLR2 antagonist (HY‐112146, MCE, USA), a TLR4 antagonist (HY‐11109, MCE, USA), a TLR9 antagonist ( HY131952 , MCE, USA), and a RAGE antagonist (HY‐P2268).

Techniques: Quantitative RT-PCR, CCK-8 Assay, Comparison, Staining, Flow Cytometry, Western Blot

Journal: PLOS Pathogens

Article Title: Trim72 is a major host factor protecting against lethal Candida albicans infection

doi: 10.1371/journal.ppat.1012747

Figure Lengend Snippet: (A and B) Primary peritoneal macrophages pretreated with rmTrim72 (1μg/ml) or vehicle were stimulated with C . albicans for the indicated periods and analyzed by Western blotting for the indicated signaling molecules. Representative images were shown (A). The intensity of the proteins was measured (B) (n = 3 per group). (C and D) WT or Trim72 -/- macrophage were stimulated with C . albicans for the indicated periods and analyzed by Western blotting for the indicated signaling molecules. Representative images were shown (C). The intensity of the proteins was measured (D) (n = 3 per group). (E) Peritoneal macrophages were stimulated with NF-κB inhibitor PDTC (25uM) and ERK1/2 inhibitor U0126 (20uM) or vehicle, followed by rmTrim72 (1μg/ml) or vehicle treatment. Migration of primary peritoneal macrophages was assessed in a transwell migration assay (n = 5 per group). Representative images were shown. Scale bar = 1000μm. (F) CCL2 levels of macrophages in (E) detected by ELISA (n = 5 per group). Data are representative of triplicate independent experiments. Statistical significance was calculated by two-tailed unpaired t-test (B, D), one-way ANOVA followed by Dunnett T3 multiple comparison test (E, F). Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The CCL2 inhibitor Bindarit (catalogue S3032, Selleck), the NF-κB pathway inhibitor PDTC (catalogue S3633, Selleck) and the ERK1/2 pathway inhibitor U0126 (catalogue S1102, Selleck) were administered intraperitoneally daily at a dose of 100mg/kg, 100mg/kg and 30mg/kg, respectively.

Techniques: Western Blot, Migration, Transwell Migration Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Comparison

Journal: PLOS Pathogens

Article Title: Trim72 is a major host factor protecting against lethal Candida albicans infection

doi: 10.1371/journal.ppat.1012747

Figure Lengend Snippet: (A) The study protocol for the administration of rmTrim72 (375μg/kg), PDTC (100mg/kg) and U0126 (30mg/kg) was shown. Mice were intravenous infected with 3×10 5 CFU of C . albicans . (B) Survival of mice in (A) (n = 12 per group). (C) C . albicans fungal load in kidneys from mice in (A) at 4 days after infection (n = 5 per group). (D) Blood urea nitrogen and serum creatinine levels in mice in (A) at 4 days after infection (n = 5 per group). (E) CD11b + F4/80 + macrophages in the kidneys of mice in (A) at 4 days after C . albicans infection by flow cytometry analysis (n = 5 per group). (F) CCL2 levels in renal tissue homogenates from mice in (A) detected by ELISA at 4 days after C . albicans infection (n = 5 per group). Survival data were collected from three independent experiments. Other data are representative of triplicate independent experiments. Statistical significance was calculated by Log-rank test (B), or one-way ANOVA followed by Dunnett’s multiple comparison test (C-F). Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The CCL2 inhibitor Bindarit (catalogue S3032, Selleck), the NF-κB pathway inhibitor PDTC (catalogue S3633, Selleck) and the ERK1/2 pathway inhibitor U0126 (catalogue S1102, Selleck) were administered intraperitoneally daily at a dose of 100mg/kg, 100mg/kg and 30mg/kg, respectively.

Techniques: Infection, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Comparison

Journal: PLOS Pathogens

Article Title: Trim72 is a major host factor protecting against lethal Candida albicans infection

doi: 10.1371/journal.ppat.1012747

Figure Lengend Snippet: (A) Trim72 levels were measured by ELISA in serum of patients with candidemia (n = 37) and healthy subjects (n = 20). (B) Trim72 levels in serum of candidemia survivors (n = 25) and candidemia non-survivors (n = 12). (C and D) Rates of phagocytosis (C) and killing (D) for live C . albicans (MOI = 1) by human monocyte-derived macrophages (hMDMs) treated with vehicle or rmTrim72 (1μg/ml) overnight (n = 5 per group). (E) Migration of hMDMs treated with vehicle or rmTrim72 (1μg/ml) in a transwell migration assay (n = 5 per group). (F) CCL2 levels detected by ELISA in hMDMs treated with vehicle or rmTrim72 (1μg/ml) overnight (n = 5 per group). (G) hMDMs were stimulated with NF-κB inhibitor PDTC (25uM) and ERK1/2 inhibitor U0126 (20uM) or vehicle, followed by rmTrim72 (1μg/ml) or vehicle treatment. Migration of hMDMs in a transwell migration assay (n = 5 per group). (H) CCL2 levels of macrophages with the indicated treatments detected by ELISA (n = 5 per group). (I) Schematic representation of the potential mechanism by which Trim72 protects against C . albicans infection. Except for clinical research (A, B), data are representative of triplicate independent experiments. Statistical significance was calculated by nonparametric Mann Whitney U test (A, B), two-tailed unpaired t-test (C-F), or one-way ANOVA followed by Dunnett’s multiple comparison test (I, H). Data are presented as mean (A, B) or mean ± SD (C-H). * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The CCL2 inhibitor Bindarit (catalogue S3032, Selleck), the NF-κB pathway inhibitor PDTC (catalogue S3633, Selleck) and the ERK1/2 pathway inhibitor U0126 (catalogue S1102, Selleck) were administered intraperitoneally daily at a dose of 100mg/kg, 100mg/kg and 30mg/kg, respectively.

Techniques: Enzyme-linked Immunosorbent Assay, Derivative Assay, Migration, Transwell Migration Assay, Infection, MANN-WHITNEY, Two Tailed Test, Comparison