Journal: Microorganisms

Article Title: MEK/ERK-Mediated Transcriptional Repression of Metabolic Genes Confers Host Defense Against Gallid alphaherpesvirus 1 Infection via Modulating Fos Nuclear Translocation

doi: 10.3390/microorganisms14071492

Figure Lengend Snippet: MEK/ERK promotes Fos expression and activation through both transcriptional regulation and post-translational modification ( A , B ) Prediction of upstream transcriptional regulators and construction of protein interaction networks for differentially expressed genes in metabolism-related pathways following treatment with BI ( A ) and PD ( B ) inhibitors. Node size is positively correlated with the network centrality (Degree value) of the gene. Node color from dark purple to light purple represents centrality from high to low. ( C , D ) RT-qPCR detection of Fos ( C ) and JUN ( D ) mRNA transcription levels after treatment with MEK/ERK inhibitors Binimetinib (BI) and PD0325901 (PD). ( E ) Homology alignment and kinase prediction analysis of Fos protein phosphorylation sites between chicken ( Gallus gallus ) and human ( Homo sapiens ). The upper panel shows phosphorylation sites predicted by GPS 6.0, with human S362 and chicken S349 being high-confidence phosphorylation sites. The lower panel shows the results of MUSCLE multiple sequence alignment. The local short sequence containing S362/S349 is completely conserved between human and chicken species. The conserved phosphorylated serine (S) site is labeled in red. * indicates completely conserved amino acid residues. ( F ) Three-dimensional structural models of human and chicken Fos predicted by AlphaFold3 and visualized with PyMOL. Ser362 and Ser349 are located within the intrinsically disordered region and fully exposed on the protein surface. The left panel shows the full-length three-dimensional structure of Fos. The right panel provides an enlarged view of the region containing the phosphorylation site, highlighting the spatial positions of human Fos Ser362 and chicken Fos Ser349. ( G ) Western blot analysis of the effects of MEK/ERK inhibitor BI and PD treatment on total and phosphorylated protein levels of ERK1/2, Fos and JUN in uninfected cells and cells exposed to ILTV. Tubulin was used as a protein loading control. + and − indicate ILTV infection and mock treatment, respectively. Data in ( C , D ) are presented as mean ± SD, n = 3. Asterisks indicate statistical significance ( p < 0.05).

Article Snippet: Two highly selective MEK1/2 inhibitors were used in this study, with the following working concentrations: Binimetinib (MCE, HY-15202, Monmouth Junction, NJ, USA) at 1 μM, and PD0325901 (MCE, HY-10254, Monmouth Junction, NJ, USA) at 0.25 μM.

Techniques: Expressing, Activation Assay, Modification, Quantitative RT-PCR, Phospho-proteomics, Sequencing, Labeling, Western Blot, Control, Infection

Journal: Microorganisms

Article Title: MEK/ERK-Mediated Transcriptional Repression of Metabolic Genes Confers Host Defense Against Gallid alphaherpesvirus 1 Infection via Modulating Fos Nuclear Translocation

doi: 10.3390/microorganisms14071492

Figure Lengend Snippet: The MEK/ERK pathway regulates Fos subcellular localization by promoting Fos phosphorylation ( A ) Nuclear-cytoplasmic fractionation combined with Western blot detection of the effects of MEK/ERK inhibitor treatment on the expression levels of phosphorylated Fos (p-Fos Ser362) and total Fos protein in cytoplasm and nucleus of uninfected/ILTV-infected cells. HSP90 was used as a cytoplasm-specific internal reference. Lamin B was used as a nucleus-specific internal reference. Tubulin was used as a whole-cell protein loading internal reference. DMSO was used as the solvent control. BI was the Binimetinib inhibitor group. PD was the PD0325901 inhibitor group. ( B ) Indirect immunofluorescence staining combined with laser confocal microscopy observation of the effects of MEK/ERK inhibitor treatment on p-Fos subcellular localization and expression. Upper panel: representative images showing p-Fos (FITC, green), nuclei (DAPI, blue) and merged fluorescence signals. Scale bar, 20 μm. Lower panel: quantitative analysis of the p-Fos nuclear translocation ratio (n ≥ 50 cells per group). * indicates statistically significant difference between groups ( p < 0.05).

Article Snippet: Two highly selective MEK1/2 inhibitors were used in this study, with the following working concentrations: Binimetinib (MCE, HY-15202, Monmouth Junction, NJ, USA) at 1 μM, and PD0325901 (MCE, HY-10254, Monmouth Junction, NJ, USA) at 0.25 μM.

Techniques: Phospho-proteomics, Fractionation, Western Blot, Expressing, Infection, Solvent, Control, Immunofluorescence, Staining, Confocal Microscopy, Fluorescence, Translocation Assay

Journal: Microorganisms

Article Title: MEK/ERK-Mediated Transcriptional Repression of Metabolic Genes Confers Host Defense Against Gallid alphaherpesvirus 1 Infection via Modulating Fos Nuclear Translocation

doi: 10.3390/microorganisms14071492

Figure Lengend Snippet: Fos acts as a core direct transcriptional repressor of metabolic genes downstream of the MEK/ERK pathway ( A ) Clustering heatmap showing the expression profile of differentially expressed metabolism-related genes in host cells after treatment with MEK/ERK inhibitors Binimetinib (BI) and PD0325901 (PD). Red indicates upregulated genes, and blue indicates downregulated genes. ( B ) PPI network analysis of differentially expressed metabolism-related genes. Node size is positively correlated with the network centrality (Degree value) of the gene. Node color from dark purple to light purple represents centrality from high to low. ( C ) RT-qPCR detection of Fos-specific siRNA knockdown efficiency. ( D ) Western blot detection of the expression efficiency of HA-tagged Fos overexpression vector. Tubulin was used as the protein loading control. ( E ) RT-qPCR detection of the effects of Fos knockdown/overexpression on the transcription levels of six core metabolic genes. ( F ) RT-qPCR detection of the effects of Fos knockdown on the transcription levels of six core metabolic genes under ILTV infection conditions. ( G ) ChIP-qPCR detection of Fos binding to the promoter regions of six core metabolic genes, with isotype control IgG as the negative control. Data in ( C , E – G ) are expressed as mean ± SD. n = 3. * indicates statistically significant difference between groups ( p < 0.05). ns indicates no statistically significant difference.

Article Snippet: Two highly selective MEK1/2 inhibitors were used in this study, with the following working concentrations: Binimetinib (MCE, HY-15202, Monmouth Junction, NJ, USA) at 1 μM, and PD0325901 (MCE, HY-10254, Monmouth Junction, NJ, USA) at 0.25 μM.

Techniques: Expressing, Quantitative RT-PCR, Knockdown, Western Blot, Over Expression, Plasmid Preparation, Control, Infection, ChIP-qPCR, Binding Assay, Negative Control