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: Cell Death & Disease

Article Title: CD146 regulates the stemness and chemoresistance of hepatocellular carcinoma via JAG2-NOTCH signaling

doi: 10.1038/s41419-025-07470-x

Figure Lengend Snippet: A , B RT-qPCR analysis of Notch signaling pathway genes in CSQT-2-Ctrl and CSQT-2-CD146 cells ( A ) or shCtrl and shCD146 cells ( B ). The protein levels of JAG2, NOTCH1 and HES1 in CSQT-2-Ctrl and CSQT-2-CD146 cells ( C ) or shCtrl and shCD146 cells ( D ). E , F RT-qPCR analysis of JAG2, NOTCH1 and HES1 in shCtrl, shCD146 cells, shCtrl with stably JAG2 overexpression, shCD146 cells with stably JAG2 overexpression. G NOTCH1 and HES1 in shCtrl, shCD146 cells, shCtrl with stably JAG2 overexpression, shCD146 cells with stably JAG2 overexpression were detected by western blotting. H JAG2, NOTCH1 and HES1 in CSQT-2-Ctrl and CSQT-2-CD146 cells treated with DMSO or QNZ(EVP4593) (NF-κB signaling inhibitor, 5 μM) for 24 h were detected by western blotting. I Western blot analysis of p65 in the cytoplasm and nucleus of CSQT-2-Ctrl and CSQT-2-CD146 cells. Data are representative of at least three independent experiments and shown as mean ± s.d. (*p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant).

Article Snippet: The Notch inhibitor RO4929097 (S1575) and NF-kB inhibitor QNZ (EVP4593) (S4902) were obtained from Selleck.

Techniques: Quantitative RT-PCR, Stable Transfection, Over Expression, Western Blot