erk 1 2 inhibitor  (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: Cancer Drug Resistance

Article Title: ZEB2 upregulation modulates the polarization of TAMs toward the immunosuppressive state in EGFR-TKI-resistant NSCLC

doi: 10.20517/cdr.2024.206

Figure Lengend Snippet: ZEB2 expression was regulated via the PI3K-Akt pathway in EGFR-TKI-resistant NSCLC. (A) The PI3K-Akt and MAPK signaling pathways were among the most significantly upregulated downstream pathways after EGFR-TKI resistance development in HCC827 and HCC4006 NSCLC cell lines. Common upregulated DEGs of these two cell lines before and after erlotinib resistance were included in this KEGG analysis; (B) The correlation between ZEB2 and key biomarkers of the PI3K-Akt [Akt1(R = 0.203), Akt2(R = 0.173), Akt3(R = 0.609)], MAPK [MAPK(R = 0.314), MAPK8(R = 0.091), MAPK14(R = 0.345)], and NF-κB signaling pathways [NF-κB(R = 0.482), p65(R = 0.169)] were assessed in the LUAD cohort from the TCGA database; (C) Western blotting was conducted to detect the phosphorylation level of key modulators of the PI3K-Akt, MAPK, and NF-κB signaling pathways before and after EGFR-TKI resistance in PC9 and HCC827 cells; (D-F) After 72 h of intervention with gradient concentrations of specific inhibitors of Akt(MK2206), ERK(U0126), and NF-κB(PDTC), western blotting was performed to determine the expression of ZEB2 in PC9-GR and HCC827-GR cells. Representative protein bands and the average results from 3 independent experiments are shown. *** P < 0.0005, **** P < 0.0001.

Article Snippet: To assess the activation of the PI3K-Akt, MAPK, and NF-κB signaling pathways, AKT1/2/3 inhibitor MK2206 (MedChemExpress, HY10358), ERK 1/2 inhibitor U0126 (MedChemExpress, HY12031A), and NF-κB inhibitor PDTC (MedChemExpress, HY18738) were used respectively on resistant NSCLC cells for 72 h. Then, the mRNA and protein of these cells were harvested for the following experiments.

Techniques: Expressing, Protein-Protein interactions, Western Blot, Phospho-proteomics

Journal: BMB Reports

Article Title: C-reactive protein accelerates DRP1-mediated mitochondrial fission by modulating ERK1/2-YAP signaling in cardiomyocytes

doi: 10.5483/BMBRep.2023-0127

Figure Lengend Snippet: Regulation of CRP-induced mitochondrial fragmentation depends on ERK1/2 phosphorylation. To determine DRP1 translocation, cells were treated with CRP for 6 h, after which the following experiments were performed: (A) Confocal immunofluorescence analysis of DRP1 (red) and Tom20 (green) visualized the mitochondrial translocation of DRP1. Scale bar: 5 μm. (B) DRP1 protein levels in cytosolic and mitochondrial fractions were quantified using GAPDH and VDAC1, respectively. (C) H9c2 cardiomyocytes were pretreated with ERK1/2 inhibitor U0126 (5 μM) to verify the ERK1/2-mediated DRP1 expression. Immunoblotting of whole cell lysates showed the total and phosphorylated forms of ERK1/2 and DRP1 expression. (D) Cytosolic and mitochondrial fractions were prepared, and protein levels were compared with or without U0126 treatment. Data represent the mean of at least five independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: U0126 (an ERK 1/2 inhibitor) was obtained from Sigma-Aldrich (St. Louis, MO, USA).

Techniques: Translocation Assay, Immunofluorescence, Expressing, Western Blot

Journal: BMB Reports

Article Title: C-reactive protein accelerates DRP1-mediated mitochondrial fission by modulating ERK1/2-YAP signaling in cardiomyocytes

doi: 10.5483/BMBRep.2023-0127

Figure Lengend Snippet: Effect of CRP on mitochondrial dynamics-related proteins regulated by ERK1/2-YAP signaling in H9c2 cardiomyocytes. H9c2 cardiomyocytes were pretreated with U0126 (5 μM) for 1 h before CRP treatment. (A) Expression levels of mitophagy-related proteins were quantified through immunoblotting. (B) To determine whether YAP mediated ERK1/2-regulated mitochondrial damage, immunoblotting was performed to determine the protein levels of YAP and TAZ. (C) Cells were transfected with siYAP (30 nM) for 4 h to determine whether YAP contributed to the changes in protein expression levels, and YAP deletion was confirmed via RT-PCR. (D) Expression of DRP1, PINK1, and PARK2 was detected through immunoblotting using each antibody, comparing with and without YAP knockdown. Data represent the mean of at least five independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: U0126 (an ERK 1/2 inhibitor) was obtained from Sigma-Aldrich (St. Louis, MO, USA).

Techniques: Expressing, Western Blot, Transfection, Reverse Transcription Polymerase Chain Reaction