stat2  (Bethyl)

Journal: Cellular & molecular immunology

Article Title: Avian influenza viruses suppress innate immunity by inducing trans-transcriptional readthrough via SSU72.

doi: 10.1038/s41423-022-00843-8

Figure Lengend Snippet: Fig. 1 TRT enhanced by avian influenza A virus infection represses genes on the complementary DNA strand. A Gene profile of the averaged normalized expression levels of 5,052 genes in A549 cells at 24 h after H1N1/H5N1/H7N9 influenza virus infection or AF treatment. The gene bodies between the TSSs and TTSs were equally sized and scaled to 60 bins, and the gene flanking regions 4 kb upstream of the TSSs and 4 kb downstream of the TTSs were divided into 100-bp windows. B Numbers of TRT genes (FC in the expression level of the TRT region (H5N1/AF) > 5) at different times after H1N1/H5N1/H7N9 infection of A549 cells. C Spearman rank linear correlation coefficient between the upregulated expression levels of the TRT region and the downregulated expression levels of TRT-influenced genes following the trans-TRT and cis-TRT patterns, respectively, at different times after H5N1/H7N9 infection in A549 cells. D Numbers of trans-TRT-influenced genes (FC in the expression level of the trans-TRT gene (H5N1/AF) > 5 and FC in the expression level of the trans-TRT-influenced gene (H1N1/H5N1) > 1.5) at different times after H5N1/H7N9 infection in A549 cells. E Functional pathway enrichment analysis of trans-TRT-influenced genes in H5N1-infected A549 cells (two-tailed P < 0.05, Benjamini–Hochberg adjusted P < 0.05). Detection of F GLS-TRT or G IL23A-TRT in A549 cells by using fluorescence in situ hybridization (FISH). A549 cells were treated with AF/H1N1/H5N1 for 24 h [DAPI nuclear staining (blue) and FISH signals obtained using a Cy3-conjugated DNA probe (red)]. The fluorescence intensity was semiquantitatively assessed using the mean fluorescence intensity (MFI) of each cell. The data are shown as the means ± SEMs. *P < 0.05, **P < 0.01. RNA-seq coverage levels of H the GLS gene, trans-TRT region of GLS, and STAT1 gene, and I the IL23A gene, trans-TRT region of IL23A, and STAT2 gene 12 h after AF/H1N1/H5N1/H7N9 treatment of A549 cells. The gene bodies and intergenic regions, as well as the gene flanking regions 2 kb upstream of the TSSs, were divided into 50-bp windows. Only exon regions are shown in this graph. RNA-seq datasets were established in duplicate

Article Snippet: Primary antibodies specific for the following proteins/peptides were used: SSU72 (1:1000; Cell Signaling Technology, cat. no. 12816) and STAT1 (1:500; Cell Signaling Technology, cat. no. 9172), STAT2 (1:500; Bethyl Laboratories, Inc., Montgomery, Texas, USA; cat. no. A303-512A-M), Flag tag (rabbit, 1:5000; MultiSciences; cat. no. LK-ab002-100), DDDDK tag (mouse, 1:5000; MBL Inc., Ottawa, ON, Canada; cat. no. M185-3 L), β-actin (1:10000; Sigma Aldrich, Saint Louis, MO, USA; cat. no. A5441), and CRISPR/Cas9 (polyclonal; Diagenode Inc., Denville, NJ, USA; cat. no. C15310258).

Techniques: Virus, Infection, Expressing, Functional Assay, Two Tailed Test, In Situ Hybridization, Staining, RNA Sequencing

Journal: Cellular & molecular immunology

Article Title: Avian influenza viruses suppress innate immunity by inducing trans-transcriptional readthrough via SSU72.

doi: 10.1038/s41423-022-00843-8

Figure Lengend Snippet: Fig. 2 TRT inhibition by CRISPR interference enhances STAT1/STAT2 expression and cell viability. RT-qPCR analysis of the A GLS-TRT gRNA and B IL23A-TRT gRNA groups at different times after infection with H5N1 (MOI = 4). RT-qPCR analysis of C STAT1 mRNA expression in the Ctrl gRNA and GLS-TRT gRNA groups and D STAT2 mRNA expression in the Ctrl gRNA and IL23A-TRT gRNA groups at different times after infection with H5N1 (MOI = 4). Western blot analysis of E STAT1 protein expression in the Ctrl gRNA and GLS-TRT gRNA groups and F STAT2 protein expression in the Ctrl gRNA and IL23A-TRT gRNA groups at different times after infection with H5N1 (MOI = 4). β-Actin expression served as the reference control. MTS cell viability assay in the G GLS-TRT gRNA and H IL23A-TRT gRNA groups at 48 h after treatment with AF or infection with H5N1 (MOI = 4). RT-qPCR analysis of viral M2 expression levels in the I GLS-TRT gRNA and J IL23A-TRT gRNA groups at 24 h after infection with H5N1 (MOI = 4). The expression levels in I and J are normalized to the Ctrl gRNA group. Each experiment was repeated at least three times. The data are shown as the means ± SEMs. *P < 0.05, **P < 0.01

Article Snippet: Primary antibodies specific for the following proteins/peptides were used: SSU72 (1:1000; Cell Signaling Technology, cat. no. 12816) and STAT1 (1:500; Cell Signaling Technology, cat. no. 9172), STAT2 (1:500; Bethyl Laboratories, Inc., Montgomery, Texas, USA; cat. no. A303-512A-M), Flag tag (rabbit, 1:5000; MultiSciences; cat. no. LK-ab002-100), DDDDK tag (mouse, 1:5000; MBL Inc., Ottawa, ON, Canada; cat. no. M185-3 L), β-actin (1:10000; Sigma Aldrich, Saint Louis, MO, USA; cat. no. A5441), and CRISPR/Cas9 (polyclonal; Diagenode Inc., Denville, NJ, USA; cat. no. C15310258).

Techniques: Inhibition, CRISPR, Expressing, Quantitative RT-PCR, Infection, Western Blot, Control, Viability Assay

Journal: Cellular & molecular immunology

Article Title: Avian influenza viruses suppress innate immunity by inducing trans-transcriptional readthrough via SSU72.

doi: 10.1038/s41423-022-00843-8

Figure Lengend Snippet: Fig. 4 TRT is reduced and lung injury is ameliorated in SSU72 transgenic mice infected with the lethal H5N1 virus. A Western blot analysis of mouse SSU72 expression in mouse lung tissues at 3 days after treatment with AF/H1N1/H5N1. β-Actin expression served as an internal control. B Numbers of TRT genes (expression of the TRT region upregulated by more than 5 compared with the AF-treated condition) in lung tissues from control (n = 5) and SSU72 transgenic mice (n = 5) at 3 days after intratracheal infection with H5N1 (106 TCID50). The relative mRNA expression ratios of C mouse STAT1 and D STAT2 in lung tissues from control (n = 8) and SSU72 transgenic mice (n = 4) at 3 days after intratracheal infection with H5N1 virus (106 TCID50). Mouse β- actin expression served as the reference control. E Kaplan–Meier survival curves for control (n = 8) and SSU72 transgenic mice (n = 10) after intratracheal infection with H5N1 (106 TCID50). F–H Control and SSU72 transgenic mice were infected with AF or H5N1 (106 TCID50) via intratracheal instillation. F Viral titers in the lungs were assessed 4 days after infection with H5N1 in control (n = 7) and SSU72 transgenic mice (n = 3). G Wet-to-dry weight ratios of the lungs of control (n = 4) and SSU72 transgenic mice (n = 4) at 3 days after infection with H5N1. H Representative images of lung pathology in control and SSU72 transgenic mice at 3 days after H5N1 infection. The lung injury scores (means ± SEMs) and numbers of infiltrating cells per microscopic field (means ± SEMs) are shown in the bar graphs. N = 100 fields for control (n = 15) and SSU72 transgenic (n = 6) mice. Bar = 100 μm. *P < 0.05 and **P < 0.01. Each experiment except for RNA-seq analysis of lungs from mice with or without H5N1 infection was repeated at least three times

Article Snippet: Primary antibodies specific for the following proteins/peptides were used: SSU72 (1:1000; Cell Signaling Technology, cat. no. 12816) and STAT1 (1:500; Cell Signaling Technology, cat. no. 9172), STAT2 (1:500; Bethyl Laboratories, Inc., Montgomery, Texas, USA; cat. no. A303-512A-M), Flag tag (rabbit, 1:5000; MultiSciences; cat. no. LK-ab002-100), DDDDK tag (mouse, 1:5000; MBL Inc., Ottawa, ON, Canada; cat. no. M185-3 L), β-actin (1:10000; Sigma Aldrich, Saint Louis, MO, USA; cat. no. A5441), and CRISPR/Cas9 (polyclonal; Diagenode Inc., Denville, NJ, USA; cat. no. C15310258).

Techniques: Transgenic Assay, Infection, Virus, Western Blot, Expressing, Control, RNA Sequencing

Journal: PLoS Pathogens

Article Title: Nuclear dengue virus NS5 antagonizes expression of PAF1-dependent immune response genes

doi: 10.1371/journal.ppat.1010100

Figure Lengend Snippet: (A) Parental A549, PAF1 KO/rescue and STAT2 KO/rescue cells were stimulated with poly(I:C) for 3 hours and subjected to RNA-seq and DESeq2 differential gene expression analysis. Results are based on three independent biological replicates. (B) Principal component analysis performed on all samples showed a clear separation between principal components (PC) describing untreated/treated cells and cell genotype. (C) GSEA was performed on genes differentially expressed in KO cells compared to parental A549 cells following poly(I:C) treatment. Up to the top 5 positively and negatively enriched Reactome pathways were plotted for each comparison (p adj < 0.1). A full list of GSEA results is available in . (D) Changes in gene expression caused by poly(I:C) treatment are shown for the subset of immune response genes (GO:0006955) significantly upregulated for poly(I:C)-treated parental A549 cells relative to mock-treated A549 cells (log2 fold change > 0.5, padj < 0.05). A Wilcoxon signed rank test with Bonferroni correction was performed to identify significant changes caused by PAF1 or STAT2 KO. (E) All genes significantly upregulated for poly(I:C)-treated parental A549 cells relative to mock-treated A549 cells (log2 fold change > 0.5, padj < 0.05) were plotted based on log2 fold change of PAF1 and STAT2 KO cells relative to parental A549 cells following poly(I:C) treatment. Parental A549 cells were used as a normalization so that it was identical for both comparisons. Unsupervised K-means clustering was also performed to identify genes with similar behavior (triangles, circles and squares). P values were adjusted for false discovery rate using the Benjamini Hochberg method. Significant changes in gene expression are plotted for PAF1 KO (cyan), STAT2 KO (magenta), both (yellow) or neither (grey). Immune response genes (GO:0006955) are highlighted with larger markers and opaque coloring.

Article Snippet: PAF1 and STAT2 KO were verified by Sanger sequencing and immunoblotting using antibodies against PAF1 (1:1000, Bethyl Labs, A300-173A) or STAT2 (1:200, Santa Cruz Biotechnology) ( ).

Techniques: RNA Sequencing, Gene Expression, Comparison

Journal: PLoS Pathogens

Article Title: Nuclear dengue virus NS5 antagonizes expression of PAF1-dependent immune response genes

doi: 10.1371/journal.ppat.1010100

Figure Lengend Snippet: (A) The protein interaction between flavivirus NS5s and PAF1 was tested biochemically. Plasmids encoding NS5s and GFP were transfected and affinity purified in HEK293T cells via a 2xStrep II tag. Immunoblot analysis of lysate and purified (Strep-AP) fractions were performed against PAF1, Strep, and GAPDH (loading/negative control). (B) Logo analysis of amino acid conservation for flavivirus NS5s from (A) in the PAF1-interacting region. The conserved stretch from amino acids 258–263 (magenta) was targeted for alanine scanning leading to the generation of 2 NS5 mutants: LGS258AAA (NS5 LGS ) and GTR261AAA (NS5 GTR ). (C) DENV2 NS5 structure from PDB (5ZQK) with PAF1-interacting region highlighted (box). The PAF1-interacting region overlaps with the C-terminal end of the MTase (yellow) and the flexible linker domain (cyan), but not the RdRP (grey). The PAF1-interacting region includes a stretch of amino acids conserved in PAF1-interacting flaviviruses tested in (A). (D) Mutants from (B) were tested for an interaction with PAF1C biochemically. Purification and immunoblot analysis were conducted as in (A). PAF1C complex members CTR9, LEO1, CDC73 and PAF1 were probed. STAT2 was used as a positive control for an NS5 interaction outside of the PAF1C-interacting region. Abbreviations: Zika virus (ZIKV), West Nile virus (WNV), Powassan virus (POWV), Langat virus (LGTV), tick-borne encephalitis virus (TBEV), Strep-affinity purified (Strep-AP).

Article Snippet: PAF1 and STAT2 KO were verified by Sanger sequencing and immunoblotting using antibodies against PAF1 (1:1000, Bethyl Labs, A300-173A) or STAT2 (1:200, Santa Cruz Biotechnology) ( ).

Techniques: Transfection, Affinity Purification, Western Blot, Purification, Negative Control, Positive Control, Virus

Journal: PLoS Pathogens

Article Title: Nuclear dengue virus NS5 antagonizes expression of PAF1-dependent immune response genes

doi: 10.1371/journal.ppat.1010100

Figure Lengend Snippet: Relative gene expression was plotted as log2 fold change for (A) PAF1 KO versus parental A549 cells or (B) STAT2 KO versus parental A549 (same as ), and NS5 mutant versus WT. Unsupervised K-means clustering was also performed to identify genes with similar behavior (triangles, circles and squares). P values were adjusted for false discovery rate using the Benjamini Hochberg method. Significant changes in gene expression are plotted for: PAF1 KO (cyan), both (yellow), neither (grey), NS5 LGS (orange), NS5 GTR (red) and NS5 2xNLS (purple). Immune response genes (GO:0006955) are highlight with larger markers and opaque coloring.

Article Snippet: PAF1 and STAT2 KO were verified by Sanger sequencing and immunoblotting using antibodies against PAF1 (1:1000, Bethyl Labs, A300-173A) or STAT2 (1:200, Santa Cruz Biotechnology) ( ).

Techniques: Gene Expression, Mutagenesis

Journal: eLife

Article Title: Multiple tumor suppressors regulate a HIF-dependent negative feedback loop via ISGF3 in human clear cell renal cancer

doi: 10.7554/eLife.37925

Figure Lengend Snippet:

Article Snippet: Antibody , STAT2 (rabbit polyclonal) , Bethyl A303-512A , , , , (1:1,000 for western blot, 1:25 for IHC).

Techniques: Western Blot, Recombinant