Journal: The Journal of General Virology

Article Title: Simian varicella virus inhibits the interferon gamma signalling pathway

doi: 10.1099/jgv.0.000925

Figure Lengend Snippet: SVV inhibits IFNγ-induced phosphorylation of STAT1. (a) BSC-1 cells were mock- or SVV-infected and 72 h later (72 h p.i.), stimulated with the indicated dose of IFNγ for 20 min and finally analysed for intracellular phosphorylated STAT1 (pSTAT1) expression by flow cytometry. The frequency of pSTAT1-positive mock- or SVV-infected BSC-1 cells is shown. Data represent average values ± sem from two independent experiments. *P<0.05, ***P<0.001 by two-way ANOVA test with Bonferroni correction. (b) Flow cytometric analysis of intracellular pSTAT1 and EGFP expression by BSC-1 cells infected with SVV expressing EGFP-ORF66 fusion protein (SVV.EGFP-66) (72 h p.i.), and control mock-infected BSC-1 cells, upon incubation with and without 1000 U ml−1 IFNγ for 20 min. Quadrants were set based on isotype control stained cells and the frequencies of cells within each quadrant are indicated. (c) Mock- and SVV.EGFP-66−infected BSC-1 cells were incubated with and without 1000 U ml−1 IFNγ for 20 min and total cell lysates were analysed by Western blotting for pSTAT1 (two bands, STAT1α=91 kDa and STAT1β=84 kDa), EGFP and β-actin protein expression. (d) The signal intensity of pSTAT1 and β-actin was quantified and the average ratio pSTAT1/β-actin from three experiments is shown. (e) Mock- and SVV.EGFP-66−infected BSC-1 cells (72 h p.i.) were stimulated with medium or 1000 U ml−1 IFNγ for 20 min in the presence or absence of phosphatase inhibitors phosSTOP (Roche) and sodium orthovanadate (Na3OV4). Intracellular phosphorylated STAT1 (pSTAT1) expression was determined by flow cytometry and presented as the median fluorescence intensity (MFI) ratio of cells stained for pSTAT1 and cells stained using isotype control antibodies. (f) Mock- and SVV.EGFP-66−infected BSC-1 cells were lysed at 72 h p.i. and probed with antibodies specific for STAT1 and β-actin by Western blotting. The arrowheads indicate bands of the appropriate size corresponding to STAT1 (two bands, STAT1α=91 kDa and STAT1β=84 kDa). (g) The signal intensity of STAT1 protein was quantified and normalized to the corresponding β-actin protein abundance (STAT1/β-actin ratio). (h–i) Flow cytometric analysis of GFP fluorescence intensity of BSC-1 cells transiently transfected to express STAT1-GFP and mock- and SVV-infected (48 h p.i.). The frequency of STAT1-GFP-expressing cells (h) and the MFI of STAT1-GFP (i) are shown. (j) Transiently transfected BSC-1 cells expressing STAT1-GFP (green) were mock- or wild-type SVV (SVV.wt)-infected (48 h p.i.), incubated with medium or 1000 U ml−1 IFNγ for 20 min and stained for pSTAT1 (red) and SVV (orange). Nuclei were counterstained with DAPI (blue). Representative confocal microscopy images from two independent experiments are shown. Magnification: 400×. (d, e, g, h) Data represent average values ± sem from three (d, e, h) and four (g) independent experiments. *P<0.05, **P<0.01, ***P<0.001 by unpaired Student’s t-test.

Article Snippet: ; 8 : 1 ratio of uninfected to SVV-infected cells) stimulated with and without IFNγ were analysed for pSTAT1 (mouse anti-pY701-STAT1; clone ST1P-11A5, ThermoFisher Scientific) and β-actin (mouse anti-β-actin; clone C4; Santa Cruz Biotechnology) by Western blotting.

Techniques: Phospho-proteomics, Infection, Expressing, Flow Cytometry, Control, Incubation, Staining, Western Blot, Fluorescence, Quantitative Proteomics, Transfection, Confocal Microscopy

Journal: Frontiers in Immunology

Article Title: Human N-Alpha-Acetyltransferase 60 Promotes Influenza A Virus Infection by Dampening the Interferon Alpha Signaling

doi: 10.3389/fimmu.2021.771792

Figure Lengend Snippet: The depletion of NAA60 increases IAV-induced pSTAT1 levels. A549 cells were transfected with 10 nM of CTRL or NAA60 siRNA for 72 h. One set of the cells was processed to confirm the depletion of NAA60 by RT-qPCR as in (not shown). The other set of the cells was infected with PR8 at an MOI of 1.0 for 0 h, 6 h, 12 h, or 24 h, and the cells were processed to detect the pSTAT1 (87 kDa), tSTAT1 (87 kDa), viral NP (56 kDa) and PDI polypeptide levels on separate blots by western blotting (A) . (B) The intensity of pSTAT1, tSTAT1 and corresponding PDI bands at 6 h, 12 h, and 24 h timepoints was quantified. First, the amount of pSTAT1 and tSTAT1 was normalized by the PDI amount at corresponding timepoints. Second, the amount of PDI-normalized pSTAT1 was normalized by the PDI-normalized tSTAT1 amount at each corresponding timepoint. Finally, the double-normalized values of pSTAT1 in CTRL siRNA transfected cells at indicated timepoint was considered 1-fold to determine its amount in NAA60 siRNA transfected cells at the corresponding timepoint. Error bars represent means ± standard deviation of at least three biological replicates; *P≤0.05. MW, molecular weight; Grey bar, CTRL; White bar, NAA60.

Article Snippet: The membrane was probed with primary antibodies: rabbit anti-NAA60 (1:1000; HPA040916, Sigma-Aldrich), rabbit anti-GFP (1:1000; #632592, Takara), mouse anti-NP (1:5000; EMD Millipore or 1:10000; NR-19868, obtained through BEI resources, NIAID, NIH), goat anti-NP (1:10000; kindly provided by Richard Webby, St Jude Children’s Research Hospital, USA), mouse anti-pSTAT1 (pY701, 1:1000; clone 14/P-STAT1; BD Biosciences), mouse anti-STAT1 (1:1000; clone 42/STAT1; BD Biosciences), mouse anti-viperin (1:1000; clone D5T2X; Cell Signaling), rabbit anti-ISG15 (1:1000; Cell Signaling), rabbit anti-IFITM3 (1:1000; Abcam), rabbit anti-acetyl histone H4-Lys8 (1:1000; Cell Signaling), mouse anti-acetyl-lysine (1:1000; AAC03, Cytoskeleton Inc) and rabbit anti-PDI (1:10000; Sigma-Aldrich) antibodies followed by the horseradish peroxidase (HRP)-conjugated anti-mouse, anti-goat, or anti-rabbit (1:5000; Life Technologies or Sigma-Aldrich) IgG antibodies.

Techniques: Transfection, Quantitative RT-PCR, Infection, Western Blot, Standard Deviation, Molecular Weight

Journal: Scientific Reports

Article Title: Infergen Stimulated Macrophages Restrict Mycobacterium tuberculosis Growth by Autophagy and Release of Nitric Oxide

doi: 10.1038/srep39492

Figure Lengend Snippet: Mtb infected macrophages were cultured with IFG. The cell lysate (cytosolic extract) was prepared and Western blotting was performed to monitor the expression of [ A ] pSTAT-3; [ B ] pPI3K; [ C ] pAkt; [ D ] Bcl-xL; [ E ] pSTAT-1. [ F,G ] NF-κB was detected using nuclear extract by EMSA and densitometry values are represented by bar diagram as a fold change (mean ± SEM). [ H ] The translocation of NF-κB from cytosol [US] to nucleus [IFG] was further validated by confocal microscopy using IFG stimulated Mtb infected macrophages. The green fluorescence indicates NF-κB marker p65 and blue signifies the nucleus staining with Hoechst dye (magnification: 100x). LPS is used as a positive control. US: infected macrophages [no IFG]; IFG: infected macrophages treated with IFG. Data are representative of two independent experiments. ***p ≤ 0.0002.

Article Snippet: Abs to pSTAT-3 (pY705) [612357], STAT-3 [610190], pSTAT-1 (pY701) [612132] and STAT-1 [610115] were procured from BD Biosciences (Bedford, MA) and iNOS [Ab3523] from Abcam (Cambridge, UK).

Techniques: Infection, Cell Culture, Western Blot, Expressing, Translocation Assay, Confocal Microscopy, Fluorescence, Marker, Staining, Positive Control

Journal: Scientific Reports

Article Title: Infergen Stimulated Macrophages Restrict Mycobacterium tuberculosis Growth by Autophagy and Release of Nitric Oxide

doi: 10.1038/srep39492

Figure Lengend Snippet: Mtb infected macrophages were cultured with IFG. The cell lysate (cytosolic extract) was prepared and Western blotting was performed to monitor the expression of [ A ] pSTAT-3; [ B ] pPI3K; [ C ] pAkt; [ D ] Bcl-xL; [ E ] pSTAT-1. [ F,G ] NF-κB was detected using nuclear extract by EMSA and densitometry values are represented by bar diagram as a fold change (mean ± SEM). [ H ] The translocation of NF-κB from cytosol [US] to nucleus [IFG] was further validated by confocal microscopy using IFG stimulated Mtb infected macrophages. The green fluorescence indicates NF-κB marker p65 and blue signifies the nucleus staining with Hoechst dye (magnification: 100x). LPS is used as a positive control. US: infected macrophages [no IFG]; IFG: infected macrophages treated with IFG. Data are representative of two independent experiments. ***p ≤ 0.0002.

Article Snippet: Abs to pSTAT-3 (pY705) [612357], STAT-3 [610190], pSTAT-1 (pY701) [612132] and STAT-1 [610115] were procured from BD Biosciences (Bedford, MA) and iNOS [Ab3523] from Abcam (Cambridge, UK).

Techniques: Infection, Cell Culture, Western Blot, Expressing, Translocation Assay, Confocal Microscopy, Fluorescence, Marker, Staining, Positive Control

Journal: Scientific Reports

Article Title: Infergen Stimulated Macrophages Restrict Mycobacterium tuberculosis Growth by Autophagy and Release of Nitric Oxide

doi: 10.1038/srep39492

Figure Lengend Snippet: It has been reported that IFG binds to IFNAR-2 and activates the tyrosine kinase Jak-1 and Tyk-2. Jak-1 induces and stimulates the phosphorylation of STAT-3 [pSTAT-3] . We investigated the role of IFG mediated signaling mechanism in activating macrophages to restrict the intracellular growth of Mtb . [1] The phosphorylation of STAT-3 is induced by Jak-1. [2] Subsequently, Akt is activated through STAT-3 along with PI3K. [3,4] This signaling translocates NF-κB into the nucleus and upregulates the IL-6 secretion, which receives a feed-back mechanism through pSTAT-3, Akt and therefore induces autophagy. Thus, ensuring the activation and survival of the Mtb infected macrophages. [5] The induction of autophagy in the Mtb infected macrophages directly involved in the killing of Mtb. [6] We propose that pSTAT-3 binds to the promoter sequence of INOS gene to produce ions in the cytosol that generates NO, a potent antimicrobial agent that may ultimately restrict the growth of Mtb . The data were validated by blocking the Akt-PI3K, iNOS and autophagy with their respective inhibitors to establish the role of autophagy in restricting the growth of Mtb by IFG stimulated macrophages. [7] Blocking of the Akt-PI3K, iNOS (NM), autophagy (3MA|CQ) with their specific inhibitors and therefore substantial reduction in the secretion of NO and autophagy, as well as increase in the survival of Mtb signifies that autophagy is directly involved in the killing of Mtb . Signifying that the function of iNOS and autophagy are interlinked. Overall, this mechanism illustrates that IFG plays a key role in controlling the survival of Mtb inside macrophages.

Article Snippet: Abs to pSTAT-3 (pY705) [612357], STAT-3 [610190], pSTAT-1 (pY701) [612132] and STAT-1 [610115] were procured from BD Biosciences (Bedford, MA) and iNOS [Ab3523] from Abcam (Cambridge, UK).

Techniques: Activation Assay, Infection, Sequencing, Blocking Assay