Journal: Free radical biology & medicine
Article Title: Hepatocyte-derived Fetuin-A promotes alcohol-associated liver disease in mice by inhibiting autophagy-lysosome degradation of TLR and M2 macrophage polarization.
doi: 10.1016/j.freeradbiomed.2024.09.011
Figure Lengend Snippet: Fig. 3. FetA is involved in the regulation of the Toll-like receptor signaling pathway. (A, B) Proteome profiling of liver tissue from EtOH-fed WT or FetA KO mice (n = 3 per group). (A) Volcano plot indicating the proteins that were differentially expressed (red, upregulated; blue, downregulated in the FetA KO group relative to the WT group) in a proteomic analysis of liver samples from EtOH-fed WT or FetA KO mice. (B) Gene Ontology (GO) enrichment analysis of the identified differentially expressed proteins was performed. (C) Prediction of protein–protein interactions using the STRING database showing two partner proteins, AHSG and TLR4. (D) Co-immunoprecipitation of FetA with TLR4 using anti-FetA or anti-TLR4. (E) Heatmap showing the differentially expressed proteins related to inflam matory signaling (n = 3 mice per group). Red indicates high relative expression, and blue indicates low relative expression. (F) The Tlr4 mRNA level in EtOH-fed mice was analyzed using qPCR. (G) Western blot analyses of TLR4 and its downstream proteins and the NLRP3 inflammasome in liver tissue (n = 3 Western blots for each protein). Relative protein level was quantitated and normalized with Actin (below panel). β-actin was used as a loading control. (H) The membrane protein of TLR4 in liver tissues of EtOH-fed mice was analyzed by Western blot. PM, plasma membrane. (I) RAW264.7 cells were cultured with medium containing 100 mM/L ethanol for 24 h and recombinant FetA protein (0, 12.5, 25, 50, 100 μg/mL) added in the last 12h. TLR4 and its downstream associated factors were detected using western blotting. (J) RAW264.7 cells were cultured with 100 mM/L ethanol for 24 h and/or 25 μg/mL FetA added in the last 12h. The mRNA levels of Tlr4 were analyzed using qPCR (n = 3 per group). (K) RAW264.7 cells were cultured with 100 mM/L ethanol for 24 h and/or 25 μg/mL FetA added in the last 12 h. Membrane protein of TLR4 in RAW264.7 cells was analyzed using flow cytometry. Cell membrane TLR4 was further quantified by mean fluorescence intensity (MFI). Data are expressed as mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. corre-sponding control. n.s., no significance.
Article Snippet: Recombinant FetA protein was purchased from MCE (HY-P70339).
Techniques: Protein-Protein interactions, Immunoprecipitation, Expressing, Western Blot, Control, Membrane, Clinical Proteomics, Cell Culture, Recombinant, Flow Cytometry, Fluorescence