Journal: Veterinary Research

Article Title: The CD2v protein of African swine fever virus inhibits macrophage migration and inflammatory cytokines expression by downregulating EGR1 expression through dampening ERK1/2 activity

doi: 10.1186/s13567-023-01239-w

Figure Lengend Snippet: CD2v downregulates EGR1 by inhibiting the activity of ERK1/2. A Heatmap of all annotated swine genes in the locomotory behavior gene set. EGR1 is highlighted in red. B EGR1 expression in PAM-GFP and PAM-CD2v macrophages for RNA-seq (***, P < 0.001; n = 3). C EGR1 expression in PAM-GFP and PAM-CD2v macrophages was evaluated by RT-qPCR with specific primers (***, P < 0.001; n = 3). D EGR1 protein levels were determined by Western blotting in PAM-GFP and PAM-CD2v macrophages. E GSEA plots of extracellular matrix organization and extracellular matrix assembly gene sets. Rank ordered by log2Foldchange of PAM-CD2v versus PAM-GFP. F Representative Western blotting bands for CD2v, EGR1, ERK1/2, phosphorylation ERK1/2, and Tublin in PAM-GFP and PAM-GFP; G PAMs were treated with ERK1/2 inhibitor FR180204 or vehicle DMSO. The expression of EGR1 and phosphorylation ERK1/2 were evaluated by Western blotting.

Article Snippet: For the EGR1 expression vector, the sequence encoding porcine EGR1 (GenBank: XM_003123974.6) flanked by BamHI and EcoRI restriction sites was synthesized and inserted into the lentiviral vector pLV-EF1a-IRES-Puro (Addgene, Cat#:85132).

Techniques: Activity Assay, Expressing, RNA Sequencing, Quantitative RT-PCR, Western Blot, Phospho-proteomics

Journal: Veterinary Research

Article Title: The CD2v protein of African swine fever virus inhibits macrophage migration and inflammatory cytokines expression by downregulating EGR1 expression through dampening ERK1/2 activity

doi: 10.1186/s13567-023-01239-w

Figure Lengend Snippet: EGR1 depleted in macrophage reduces cell migration . A Schematic illustration of EGR gene locus of swine and small guide RNA location. The EGR1 expression was determined by Western blotting in EGR1-WT PAMs and EGR1-KO1 PAMs. B Representative images of EGR1-WT and EGR1-KO1 PAMs migration at 24 h and 48 h. Scale bar: 200 μm. C The number of migratory cells from the transwell assay was evaluated by five random fields’ relative staining areas (*** P < 0.001; n = 5). D Representative images of EGR1-WT and EGR1-KO1 PAMs at 12 and 24 h after initial scratch. E Quantification of scratch width EGR1-WT and EGR1-KO1 PAMs at different time points as indicated (** P < 0.01; *** P < 0.001; ns, not significant; n = 6). F Cell proliferation of EGR1-WT and EGR1-KO1 PAMs was determined by resazurin at different time points as indicated (ns, not significant; n = 8).

Article Snippet: For the EGR1 expression vector, the sequence encoding porcine EGR1 (GenBank: XM_003123974.6) flanked by BamHI and EcoRI restriction sites was synthesized and inserted into the lentiviral vector pLV-EF1a-IRES-Puro (Addgene, Cat#:85132).

Techniques: Migration, Expressing, Western Blot, Transwell Assay, Staining

Journal: Veterinary Research

Article Title: The CD2v protein of African swine fever virus inhibits macrophage migration and inflammatory cytokines expression by downregulating EGR1 expression through dampening ERK1/2 activity

doi: 10.1186/s13567-023-01239-w

Figure Lengend Snippet: EGR1 overexpression in PAM-CD2v restores the ability of cell migration . A The EGR1 protein was determined by Western blotting in PAM-CD2v-EGR1 and PAM-CD2v-GFP cells. B Representative images of cell migration from PAM-CD2v-EGR1 and PAM-CD2v-GFP cells at 24 h and 48 h. C The number of migratory cells from the transwell assay was evaluated by five random fields’ relative staining areas (*** P < 0.001; n = 5). D Representative images of PAM-CD2v-EGR1 and PAM-CD2v-GFP cells at 12 and 24 h after initial scratch. E Quantification of scratch width of PAM-CD2v-EGR1 and PAM-CD2v-GFP cells at different time points as indicated (** P < 0.01; *** P < 0.001; ns, not significant; n = 6). F Cell proliferation of PAM-CD2v-EGR1 and PAM-CD2v-GFP were determined by resazurin at different time points as indicated (ns, not significant; n = 8).

Article Snippet: For the EGR1 expression vector, the sequence encoding porcine EGR1 (GenBank: XM_003123974.6) flanked by BamHI and EcoRI restriction sites was synthesized and inserted into the lentiviral vector pLV-EF1a-IRES-Puro (Addgene, Cat#:85132).

Techniques: Over Expression, Migration, Western Blot, Transwell Assay, Staining

Journal: Veterinary Research

Article Title: The CD2v protein of African swine fever virus inhibits macrophage migration and inflammatory cytokines expression by downregulating EGR1 expression through dampening ERK1/2 activity

doi: 10.1186/s13567-023-01239-w

Figure Lengend Snippet: ChIP-Seq analysis of genome-wide binding sites of EGR1 in PAMs . A Distribution of EGR1-binding and H3K27ac enrichment sites in PAMs. Chromatin immunoprecipitation was conducted followed by deep sequencing (ChIP-seq) with the use of an EGR1 antibody and H3K27ac antibody. The total number of EGR1-bound and H3K27ac enrichment peaks was identified, and the percentage found within various genome regions was calculated. B Profile plots and heatmaps of the distribution of EGR1-binding and H3K27ac from ± 3 kb to genes’ transcriptional start site (TSS) are shown. The x-axis denotes the position from the TSS, and the y-axis shows the signal strength of 30 440 swine genes. C Overlayed profile plot of EGR1 and H3K27ac ChIP-seq read distribution within 3 kb of TSS. D Quantitative correlation of signal strength of EGR1 binding and H3K27ac enrichment at all genes TSSs. The linear regression trendline and the correlation coefficient-square are displayed.

Article Snippet: For the EGR1 expression vector, the sequence encoding porcine EGR1 (GenBank: XM_003123974.6) flanked by BamHI and EcoRI restriction sites was synthesized and inserted into the lentiviral vector pLV-EF1a-IRES-Puro (Addgene, Cat#:85132).

Techniques: ChIP-sequencing, Genome Wide, Binding Assay, Chromatin Immunoprecipitation, Sequencing

Journal: Veterinary Research

Article Title: The CD2v protein of African swine fever virus inhibits macrophage migration and inflammatory cytokines expression by downregulating EGR1 expression through dampening ERK1/2 activity

doi: 10.1186/s13567-023-01239-w

Figure Lengend Snippet: EGR1 binds at the promoter regions of cell locomotion-related genes . Screenshot from IGV showing DNA sequence tag densities at gene APP ( A ), ID2 ( B ), and EPHA4 ( C ) locus following ChIP-seq with the indicated antibodies in PAMs. The maximum coverage for each track is shown on the left. D Relative expression levels of gene APP, EPHA4, and ID2 in PAM-GFP and PAM-CD2v (** P < 0.01; *** P < 0.001; n = 3). E Relative expression levels of gene APP, EPHA4, and ID2 in EGR1-WT and EGR1-KO PAMs (*** P < 0.001; n = 3). (F) Relative expression levels of gene APP, EPHA4, and ID2 in PAM-CD2v-EGR1 and PAM-CD2v-GFP (** P < 0.01; n = 3).

Article Snippet: For the EGR1 expression vector, the sequence encoding porcine EGR1 (GenBank: XM_003123974.6) flanked by BamHI and EcoRI restriction sites was synthesized and inserted into the lentiviral vector pLV-EF1a-IRES-Puro (Addgene, Cat#:85132).

Techniques: Sequencing, ChIP-sequencing, Expressing

Journal: Veterinary Research

Article Title: The CD2v protein of African swine fever virus inhibits macrophage migration and inflammatory cytokines expression by downregulating EGR1 expression through dampening ERK1/2 activity

doi: 10.1186/s13567-023-01239-w

Figure Lengend Snippet: CD2v dampens the response to the ERK1/2 pathway through EGR1. A The Venn diagram illustrates the overlapping of EGR1 (shown in blue) and H3K27ac (shown in red) binding peaks. B The Venn diagram demonstrates the overlapped genes that are co-occupied by EGR1 and H3K27ac (black), genes containing EGR1 binding peaks within 1 kb up or downstream of their TSS (red), genes containing H3K27ac binding peaks within 1 kb up or downstream of their TSS (blue), and differential expressed genes (adjust p < 0.05 and log 2 FoldChange < 0) in PAM-CD2v versus PAM-GFP (green). C GO term enrichment analysis of 247 overlapped genes from B.

Article Snippet: For the EGR1 expression vector, the sequence encoding porcine EGR1 (GenBank: XM_003123974.6) flanked by BamHI and EcoRI restriction sites was synthesized and inserted into the lentiviral vector pLV-EF1a-IRES-Puro (Addgene, Cat#:85132).

Techniques: Binding Assay

Journal: Veterinary Research

Article Title: The CD2v protein of African swine fever virus inhibits macrophage migration and inflammatory cytokines expression by downregulating EGR1 expression through dampening ERK1/2 activity

doi: 10.1186/s13567-023-01239-w

Figure Lengend Snippet: CD2v inhibits the expression of inflammatory cytokines in swine macrophages. Various derivatives of PAM were treated with 2 μg/mL LPS for 4 h. The expression levels of inflammatory cytokine genes were evaluated by qPCR. A Relative expression levels of gene IL-1α, IL-1β, IL6, IL8, and TNFα in PAM-GFP and PAM-CD2v (*** P < 0.001; n = 4). B Relative expression levels of gene IL-1α, IL-1β, IL6, IL8, and TNFα in EGR1-WT and EGR1-KO PAMs (** P < 0.01; *** P < 0.001; n = 4). C Relative expression levels of gene IL-1α, IL-1β, IL6, IL8, and TNFα in PAM-CD2v-EGR1 and PAM-CD2v-GFP (** P < 0.01; *** P < 0.001; n = 4).

Article Snippet: For the EGR1 expression vector, the sequence encoding porcine EGR1 (GenBank: XM_003123974.6) flanked by BamHI and EcoRI restriction sites was synthesized and inserted into the lentiviral vector pLV-EF1a-IRES-Puro (Addgene, Cat#:85132).

Techniques: Expressing