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

Journal: PLoS Pathogens

Article Title: Transposon-mediated Chromosomal Integration of Transgenes in the Parasitic Nematode Strongyloides ratti and Establishment of Stable Transgenic Lines

doi: 10.1371/journal.ppat.1002871

Figure Lengend Snippet: A ) Donor vector pPV254 incorporating a fluorescent reporter gene in which expression of GFP is driven by the Ss-act-2 promoter and terminated by the Ss era-1 3′ UTR. The reporter transgene in pPV254 is flanked by the inverted terminal repeats (ITR) plus internal sequences common to piggyBac transposable elements. B ) Donor vector pPV356, which is like pPV254 in all respects except that the coding sequence is flanked by the gypsy retroviral insulator sequences from Drosophila . C ) Helper vector pPV402 in which expression of the piggyBac transposase gene is driven by Ss-rps-21 promoter and terminated by the Ss-era-1 3′ UTR. D ) Plasmid pPV257 for in vitro transcription of mRNA encoding the piggyBac transposase under the T7 promoter. In lieu of helper vector pPV402, this mRNA was capped, tailed and co-injected with donor vector pPV356 in Experiment 2.

Article Snippet: Finally, the expression cassette, Ss-act-2 p:: gfp :: Ss-era-1 3′UTR from pAJ08 was cloned into the MCS by digestion with Xba I and Hind III and ligation with T4 DNA Ligase to yield pPV356. pPV402 ( ; GenBank accession number, JX013634), the piggyBac transposase helper vector, was made by excising the piggyBac transposase coding sequence from pPV257 (see below) with the restriction enzymes AgeI and AvrII and cloning them into the pAJ50 vector (Addgene Plasmid #14918) from which the gfp coding region had been removed with the same restriction enzymes.

Techniques: Plasmid Preparation, Expressing, Sequencing, Retroviral, In Vitro, Injection

Journal: PLoS Pathogens

Article Title: Transposon-mediated Chromosomal Integration of Transgenes in the Parasitic Nematode Strongyloides ratti and Establishment of Stable Transgenic Lines

doi: 10.1371/journal.ppat.1002871

Figure Lengend Snippet: Heritable transgene expression and establishment of stable transgene-expressing lines in S. ratti as a function of various pairings of donor and helper plasmids incorporating elements of the piggyBac transposon.

Article Snippet: Finally, the expression cassette, Ss-act-2 p:: gfp :: Ss-era-1 3′UTR from pAJ08 was cloned into the MCS by digestion with Xba I and Hind III and ligation with T4 DNA Ligase to yield pPV356. pPV402 ( ; GenBank accession number, JX013634), the piggyBac transposase helper vector, was made by excising the piggyBac transposase coding sequence from pPV257 (see below) with the restriction enzymes AgeI and AvrII and cloning them into the pAJ50 vector (Addgene Plasmid #14918) from which the gfp coding region had been removed with the same restriction enzymes.

Techniques: Expressing

Journal: PLoS Pathogens

Article Title: Transposon-mediated Chromosomal Integration of Transgenes in the Parasitic Nematode Strongyloides ratti and Establishment of Stable Transgenic Lines

doi: 10.1371/journal.ppat.1002871

Figure Lengend Snippet: Southern hybridization of genomic DNA (gDNA) of S. ratti from stably transformed lines probed for the gfp coding sequence. A ) Transgene diagram showing position of the probe used for Southern hybridization analysis and the single restriction site for BsrGI, the enzyme used for restriction digestion of gDNA. B ) Southern blot of BsrGI digests of gDNA from pooled free-living adults from three independent integrated lines (PV2, PV3 and PV4). The positive control lane is a blot of a BsrGI digest of donor plasmid pPV356, and the negative control is a blot of a BsrGI digest of gDNA from non-transformed S. ratti free-living adults ( S.r. gDNA). Note gfp hybridization signals in multiple restriction fragments in gDNA from each of the three integrated lines. A single hybridizing band of 8.1 kb appears as predicted in the positive control digest of pPV356. No gfp -specific signal is detected in the negative control digest of gDNA from non-transformed S. ratti . C ) Gel analysis of PCR products from genomic DNA templates from non-transformed control parasites, parasites from each of three stable lines, PV2, PV3 and PV4, with integrated, stably expressed transgenes and F1 progeny of free-living female worms microinjected with donor plasmid pPV356 and helper plasmid pPV402. Also included are PCR products from control reactions with plasmids pPV356 and pPV254 as templates and from a reaction from which template was omitted. Upper gel image depicts 624 bp amplification products resulting from a forward primer hybridizing to the M13 reverse priming site in the vector and a reverse primer hybridizing within the transposon sequence. Lower gel image is a loading control showing the expected 554 bp amplification product from reactions with primers specific for the constitutively expressed cellular actin-encoding gene Sr-act-2 .

Article Snippet: Finally, the expression cassette, Ss-act-2 p:: gfp :: Ss-era-1 3′UTR from pAJ08 was cloned into the MCS by digestion with Xba I and Hind III and ligation with T4 DNA Ligase to yield pPV356. pPV402 ( ; GenBank accession number, JX013634), the piggyBac transposase helper vector, was made by excising the piggyBac transposase coding sequence from pPV257 (see below) with the restriction enzymes AgeI and AvrII and cloning them into the pAJ50 vector (Addgene Plasmid #14918) from which the gfp coding region had been removed with the same restriction enzymes.

Techniques: Hybridization, Stable Transfection, Transformation Assay, Sequencing, Southern Blot, Positive Control, Plasmid Preparation, Negative Control, Control, Amplification