Journal: Frontiers in Immunology

Article Title: LPS-induced extracellular AREG triggers macrophage pyroptosis through the EGFR/TLR4 signaling pathway

doi: 10.3389/fimmu.2025.1549749

Figure Lengend Snippet: EGFR inhibition and TLR4 silencing impair extracellular AREG-induced IκB phosphorylation and NFκB activation in BMDMs. Structural features of the AREG protein were analyzed using DOG2.0 software (A) . Purification of extracellular AREG was performed via Coomassie Blue staining (B) . EGFR and TLR4 expression was detected in extracellular AREG-stimulated BMDM via Immunofluorescence (C) . p-EGFR, TLR4, p-P65, and p-IκB expression levels were detected in inhibitor of EGFR (1 mM) pretreating BMDM for 4 h through Western blot (D, E) . RAW264.7 were collected for lysis after treatment with His-AREG for 3 h. Immunoprecipitation was performed with a specific antibody against the His tag, EGFR, and TLR4 to assess the interaction between extracellular AREG and EGFR (F) . p-P65 and p-Iκb expression levels were detected in TLR4 −/− BMDM and RAGE −/− BMDM using Western blot (G, H) . Data are presented as mean ± SEM (n ≥ 3). * P < 0.05, ** P < 0.01, *** P < 0.001 vs. Control. AREG, amphiregulin; BMDM, bone marrow-derived macrophages; EGFR, epidermal growth factor receptor.

Article Snippet: After the reaction, cells were incubated with primary antibodies against EGFR (ABclonal, #A23381), TLR4 (Immunoway, #YT0744), or ASC (CST, #67824).

Techniques: Inhibition, Phospho-proteomics, Activation Assay, Software, Purification, Staining, Expressing, Immunofluorescence, Western Blot, Lysis, Immunoprecipitation, Control, Derivative Assay

Journal: Frontiers in Immunology

Article Title: LPS-induced extracellular AREG triggers macrophage pyroptosis through the EGFR/TLR4 signaling pathway

doi: 10.3389/fimmu.2025.1549749

Figure Lengend Snippet: EGFR inhibition and TLR4 silencing impair AREG-induced macrophage pyroptosis. BMDM was stimulated with AREG+ATP or LPS+ATP, and the expression of NLRP3, p-P65, p-IκB, CASPASE-1-p20, and GSDMD-N was detected via Western blot (A-C) . BMDM was stimulated with AREG+ATP or LPS+ATP, and oligomerization of ASC was detected using immunofluorescence (D, E) . Experimental diagram of AREG-induced macrophage pyroptosis. For the priming step, BMDM was treated with AREG for 2.5 as the first signal and the ATP as the second signal (F) . NLRP3, CASPASE-1-p20, and GSDMD-N expressions were detected in the EGFR inhibitor (1 mM) pretreating AREG +ATP-induced BMDM for 4 h through Western blot (G, J) . NLRP3, CASPASE-1-p20, and GSDMD-N expressions were detected in AREG +ATP-induced TLR4 −/− BMDM via Western blot (H, K) . The expression of IL-1b and IL-18 was detected in the supernatant of AREG +ATP-induced TLR4 −/− BMDM via ELISA (I) . Data are presented as mean ± SEM (n ≥ 3). * P < 0.05, ** P < 0.01, *** P < 0.001 vs. Control. ns, no significant; AREG, amphiregulin; BMDM, bone marrow-derived macrophages; EGFR, epidermal growth factor receptor; GSDMD, gasdermin D; ATP, adenosine triphosphate.

Article Snippet: After the reaction, cells were incubated with primary antibodies against EGFR (ABclonal, #A23381), TLR4 (Immunoway, #YT0744), or ASC (CST, #67824).

Techniques: Inhibition, Expressing, Western Blot, Immunofluorescence, Enzyme-linked Immunosorbent Assay, Control, Derivative Assay

Journal: Frontiers in Immunology

Article Title: LPS-induced extracellular AREG triggers macrophage pyroptosis through the EGFR/TLR4 signaling pathway

doi: 10.3389/fimmu.2025.1549749

Figure Lengend Snippet: Neutralizing extracellular AREG decreases LPS-induced TLR4 expression and pyroptosis in macrophages. LPS-induced BMDM was pretreated with a neutralizing antibody of AREG. p-EGFR, TLR4, and GSDMD-N expression levels were detected via Western blot and immunofluorescence (A-C, E, F) , TLR4 expression and ASC oligomerization was detected through immunofluorescence (D, G, H) . Formation of pyrosomes (red arrows) was detected using electron microscopy, scale bars, 2 μm (I) . Data are presented as mean ± SEM (n ≥ 3).* P < 0.05, ** P < 0.01, *** P < 0.001 vs. Control. AREG, amphiregulin.

Article Snippet: After the reaction, cells were incubated with primary antibodies against EGFR (ABclonal, #A23381), TLR4 (Immunoway, #YT0744), or ASC (CST, #67824).

Techniques: Expressing, Western Blot, Immunofluorescence, Electron Microscopy, Control

Journal: Frontiers in Immunology

Article Title: LPS-induced extracellular AREG triggers macrophage pyroptosis through the EGFR/TLR4 signaling pathway

doi: 10.3389/fimmu.2025.1549749

Figure Lengend Snippet: Schematic diagram illustrating how AREG triggers macrophage pyroptosis through the EGFR/TLR4 signaling pathway during inflammatory responses. Schematic diagram was created in https://BioRender.com . Mechanistically, extracellular AREG expression is regulated by the translational regulation of RPLP1. When extracellular AREG and ATP jointly stimulate macrophages, AREG promotes TLR4 expression by binding to EGFR. Expression of TLR4 recruits the adaptor protein Myd88 and further activates downstream IκB and NFκB, which promotes the NLRP3 inflammasome and subsequent pyroptosis. AREG, amphiregulin; EGFR, epidermal growth factor receptor; ATP, adenosine triphosphate.

Article Snippet: After the reaction, cells were incubated with primary antibodies against EGFR (ABclonal, #A23381), TLR4 (Immunoway, #YT0744), or ASC (CST, #67824).

Techniques: Expressing, Binding Assay

Journal: Discover oncology

Article Title: Exploring the combined roles of GALNT1 and GALNT2 in hepatocellular carcinoma malignancy and EGFR modulation.

doi: 10.1007/s12672-025-02069-2

Figure Lengend Snippet: Fig. 6 GALNT1/ GALNT2 modifies the activity of EGFR. A expression of EGFR in liver cell lines examined. B, C EGFR protein expression after transfection of GALNT1 and GALNT2 in hepatocellular carcinoma cell lines detected. D protein levels of EGFR after MHCC97L and Huh7 cells were transfected with sh-NC, shGALNT1, si-NC, siGALNT2, pcNC, pcGALNT1, pcGALNT2, shG1 + siG2, shG1 + pcG2, pcG1 + siG2or pcG1 + pcG2. All experiments were conducted in triplicate and the results are presented as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 , ****P < 0.0001 and ns. indicates non significance

Article Snippet: After blocking the membranes with 5% skim milk, primary antibodies against EGFR (1:1000, Proteintech), GAPDH (1:2000, ABclone), GALNT1 (1:1000, Invitrogen), and GALNT2 (1:1000, ABclone) were added.

Techniques: Activity Assay, Expressing, Transfection

Journal: Discover oncology

Article Title: Exploring the combined roles of GALNT1 and GALNT2 in hepatocellular carcinoma malignancy and EGFR modulation.

doi: 10.1007/s12672-025-02069-2

Figure Lengend Snippet: Fig. 7 A graphical illustra- tion depicting the impact of GALNT1 and GALNT2 on EGFR expression in HCC was created using BioRender.com

Article Snippet: After blocking the membranes with 5% skim milk, primary antibodies against EGFR (1:1000, Proteintech), GAPDH (1:2000, ABclone), GALNT1 (1:1000, Invitrogen), and GALNT2 (1:1000, ABclone) were added.

Techniques: Expressing

Journal: Frontiers in Pharmacology

Article Title: Oxyresveratrol as a novel ferroptosis inducer exhibits anticancer activity against breast cancer via the EGFR/PI3K/AKT/GPX4 signalling axis

doi: 10.3389/fphar.2024.1527286

Figure Lengend Snippet: ORes induces ferroptosis in breast cancer cells via the EGFR/PI3K/AKT/GPX4 signalling axis. (A) The table indicates the top 10 connectivity scores between the input gene signature and the gene signatures of compounds in the LINCS L1000 touchstone dataset. (B) The table displays the top 10 scores between the input gene signature and the gene signatures of compounds in the DGDB dataset. (C) GSEA of HTS 2 results of Osimertinib dimesylate–treated MDA-MB-231 cells. (D) GSEA of HTS 2 results of AZ-5104–treated MDA-MB-231 cells. (E) Volcano plot of the DEGs in EGFR knockdown MDA-MB-231 cells, with red and blue dots indicating upregulated and downregulated genes, respectively. Differential gene screening criteria: |FoldChange|>1.3 and p- value < 0.05. (F) Representative Western blot results. (G) Quantification of western blots. Experiments were performed in triplicate, and data are presented as mean ± SD. *** p < 0.001; ns, no significance.

Article Snippet: The membranes were incubated overnight at 4°C with primary antibodies against GPX4 (Cell Signaling Technology, 59735, 1:1,000), phosphorylated EGFR (ZenBio, R26283, 1:1,000), EGFR (Selleck, A5858, 1:1,000), phosphorylated PI3K (ZenBio, 310164, 1:1,000), PI3K (ZenBio, 200900, 1:1,000), phosphorylated AKT (Cell Signaling Technology, 13038, 1:1,000), and AKT (Cell Signaling Technology, 4691, 1:1,000).

Techniques: Knockdown, Western Blot