Journal: medRxiv

Article Title: Polymorphism in IFNAR contributes to glucocorticoid response and outcome in ARDS and COVID-19

doi: 10.1101/2022.03.10.22272123

Figure Lengend Snippet: (A ) STAT1 expression in the lung after 4-day culture in the presence of IFN beta with or without hydrocortisone (HC). ( B) pSTAT1 expression in the same specimens as in A. ( C) Example photomicrographs showing higher STAT2 expression in a TT patient than in a CT patient and the effect of HC on its nuclear translocation. Most STAT2 remains in the cytoplasm of the CT patients, whereas nuclear expression is prominent in the TT patient. Indicated insets are shown in the bottom row. Arrows. ( D ) Combined results of all patients noting that two CT samples are excluded in the data as the patients were already under glucocorticoid treatment at the time of sample acquisition. Ns, not significant; *P<0.05; **P<0.01; and ***P<0.001

Article Snippet: The first stage antibodies were anti-alpha chain of the IFN alpha/beta receptor (St John’s Laboratory STJ112765) that was used 1:2000 and 1:5000 and anti-Stat1 (1:400, 9175S), anti-pStat1(1:100, 9167S) and anti-Stat2 (1:200, 72604S) all from Cell Signalling.

Techniques: Expressing, Translocation Assay

Journal: PLoS Pathogens

Article Title: Human Cytomegalovirus IE1 Protein Elicits a Type II Interferon-Like Host Cell Response That Depends on Activated STAT1 but Not Interferon-γ

doi: 10.1371/journal.ppat.1002016

Figure Lengend Snippet: A) Specific reduction in STAT1 (left) and STAT2 (right) protein levels by siRNA-mediated gene silencing. MRC-5 cells were transfected with the indicated siRNA duplexes. Two and five days post transfection, whole cell protein extracts were prepared and subjected to immunoblotting with anti-STAT1α, anti-STAT2, and anti-GAPDH antibodies. B) STAT1 (left) but not STAT2 (right) knock-down abolishes IE1-mediated ISG induction. TetR and TetR-IE1 cells were transfected with the indicated siRNA duplexes. Two days post transfection, cells were treated with doxycycline for 72 h. Relative mRNA expression levels were determined by qRT-PCR with primers specific for the CXCL10, GBP4, IE1, STAT1, and STAT2 genes. Results were normalized to TUBB and mean values with standard deviations from two biological and two technical replicates are shown. CXCL10, GBP4, STAT1, and STAT2 expression is shown in comparison to control siRNA-transfected TetR cells (set to 1). IE1 expression is presented relative to control siRNA-transfected TetR-IE1 cells (set to 1).

Article Snippet: Immunodetection employed primary mono- or polyclonal antibodies directed against hCMV IE1 (1B12; ) or human GAPDH (Abcam, no. ab9485), histone H2A (Abcam, no. ab13923), STAT1 (no. sc-464 for immunoblotting and no. sc-346 for immunofluorescence, both from Santa Cruz), STAT1α (Santa Cruz, no. sc-345), STAT2 (Santa Cruz, no. sc-22816), and phosphorylated STAT1 (Y701-specific antibody no. 9171 and S727-specific antibody no. 9177, both from Cell Signaling Technologies).

Techniques: Transfection, Western Blot, Knockdown, Expressing, Quantitative RT-PCR, Comparison, Control

Journal: PLoS Pathogens

Article Title: Human Cytomegalovirus IE1 Protein Elicits a Type II Interferon-Like Host Cell Response That Depends on Activated STAT1 but Not Interferon-γ

doi: 10.1371/journal.ppat.1002016

Figure Lengend Snippet: A) IE1 expression leads to increased steady-state levels of Y701- and S727-phosphorylated STAT1. TetR and TetR-IE1 cells were treated for 72 h with doxycycline and for 1 h with solvent (–) or IFN-γ. Whole cell protein extracts were prepared and subjected to immunoblotting with anti-STAT1, anti-pSTAT1 (Y701), anti-pSTAT1 (S727), anti-GAPDH, and anti-IE1 antibodies. B) Verification of knock-down resistance and phosphorylation deficiency of STAT1 variants. TetR-IE1 cells without (–) and with stable expression of ectopic wild-type STAT1 (STAT1), siRNA-resistant wild-type STAT1 (STAT1*), and siRNA-resistant phosphorylation-deficient STAT1 (STAT1*Y701F and STAT1*S727A) were transfected with negative control (#149) or STAT1-specific (#146) siRNA duplexes. Two days post transfection cells were treated for 1 h with IFN-γ. Whole cell protein extracts were prepared and subjected to immunoblotting with anti-STAT1, anti-pSTAT1 (Y701), anti-pSTAT1 (S727), and anti-GAPDH antibodies. C) Ectopic wild-type STAT1 but not phosphorylation-deficient STAT1 mutants efficiently rescue IE1-dependent ISG induction in cells depleted of endogenous STAT1. TetR-IE1 cells without (–) and with stable expression of the indicated ectopic STAT1s were transfected with control (#149) or STAT1-specific (#146) siRNA duplexes. Two days post transfection cells were treated for 72 h with doxycycline. Relative mRNA expression levels were determined by qRT-PCR with primers specific for the CXCL10, GBP4, IE1, and STAT1 genes. Results were normalized to TUBB and mean values with standard deviations from two biological and two technical replicates are shown. Expression is shown in comparison to control siRNA-transfected TetR-IE1 cells without ectopic STAT1 expression (set to 1).

Article Snippet: Immunodetection employed primary mono- or polyclonal antibodies directed against hCMV IE1 (1B12; ) or human GAPDH (Abcam, no. ab9485), histone H2A (Abcam, no. ab13923), STAT1 (no. sc-464 for immunoblotting and no. sc-346 for immunofluorescence, both from Santa Cruz), STAT1α (Santa Cruz, no. sc-345), STAT2 (Santa Cruz, no. sc-22816), and phosphorylated STAT1 (Y701-specific antibody no. 9171 and S727-specific antibody no. 9177, both from Cell Signaling Technologies).

Techniques: Expressing, Solvent, Western Blot, Knockdown, Phospho-proteomics, Transfection, Negative Control, Control, Quantitative RT-PCR, Comparison

Journal: PLoS Pathogens

Article Title: Human Cytomegalovirus IE1 Protein Elicits a Type II Interferon-Like Host Cell Response That Depends on Activated STAT1 but Not Interferon-γ

doi: 10.1371/journal.ppat.1002016

Figure Lengend Snippet: A) TetR and TetR-IE1 cells were treated with doxycycline for 72 h. Where indicated, TetR cells were incubated in the presence of IFN-γ or IFN-α for 1 h before samples were fixed with paraformaldehyde and examined by indirect immunofluorescence coupled to confocal microscopy. Samples were simultaneously reacted with rabbit polyclonal antibodies against STAT1 (left) or STAT2 (right) and a mouse monoclonal antibody against IE1, followed by incubation with a rabbit-specific Alexa Fluor 546 conjugate and a mouse-specific Alexa Fluor 633 conjugate. TetRnlsEGFP (TetR) fluorescence is shown to visualize nuclei. Additionally, merge images of STAT, IE1, and TetR signals are presented. Scale bar, 10 µm. B) TetR-IE1 cells were treated with doxycycline for 0 h, 24 h, or 72 h. Cytoplasmic and nuclear extracts were prepared and subjected to immunoblotting with anti-STAT1, anti-STAT2, anti-GAPDH, anti-H2A, and anti-IE1 antibodies. For the right panel, TetR-IE1 cells were treated with IFN-α or IFN-γ for 1 h before fractionation.

Article Snippet: Immunodetection employed primary mono- or polyclonal antibodies directed against hCMV IE1 (1B12; ) or human GAPDH (Abcam, no. ab9485), histone H2A (Abcam, no. ab13923), STAT1 (no. sc-464 for immunoblotting and no. sc-346 for immunofluorescence, both from Santa Cruz), STAT1α (Santa Cruz, no. sc-345), STAT2 (Santa Cruz, no. sc-22816), and phosphorylated STAT1 (Y701-specific antibody no. 9171 and S727-specific antibody no. 9177, both from Cell Signaling Technologies).

Techniques: Incubation, Immunofluorescence, Confocal Microscopy, Fluorescence, Western Blot, Fractionation

Journal: PLoS Pathogens

Article Title: Human Cytomegalovirus IE1 Protein Elicits a Type II Interferon-Like Host Cell Response That Depends on Activated STAT1 but Not Interferon-γ

doi: 10.1371/journal.ppat.1002016

Figure Lengend Snippet: TetR and TetR-IE1 cells were treated with doxycycline for 72 h. During the last 30 min of doxycycline treatment TetR cells were incubated in the presence of solvent, IFN-γ or IFN-α. ChIP assays were carried out with polyclonal rabbit antibodies against STAT1 (A) or STAT2 (B). The fraction of immunoprecipitated DNA relative to input DNA was determined by qPCR with primers specific for the non-ISGs GAPDH (white circles), ribosomal protein L30 (RPL30) (black circles), and TUBB (gray circles) as well as for the ISGs GBP4 (white squares), CXCL9 (black squares), TAP1 (gray squares), IFIT2 (white triangles), and OAS1 (black triangles). Mean values of two technical replicates from TetR-IE1 cells (IE1) and from IFN-γ- or IFN-α-treated TetR cells are presented relative to solvent-treated TetR cells (set to 1). Results from five (A) or two (B) independent experiments are shown.

Article Snippet: Immunodetection employed primary mono- or polyclonal antibodies directed against hCMV IE1 (1B12; ) or human GAPDH (Abcam, no. ab9485), histone H2A (Abcam, no. ab13923), STAT1 (no. sc-464 for immunoblotting and no. sc-346 for immunofluorescence, both from Santa Cruz), STAT1α (Santa Cruz, no. sc-345), STAT2 (Santa Cruz, no. sc-22816), and phosphorylated STAT1 (Y701-specific antibody no. 9171 and S727-specific antibody no. 9177, both from Cell Signaling Technologies).

Techniques: Incubation, Solvent, Immunoprecipitation

Journal: International Journal of Molecular Sciences

Article Title: Butyrate Prevents Induction of CXCL10 and Non-Canonical IRF9 Expression by Activated Human Intestinal Epithelial Cells via HDAC Inhibition

doi: 10.3390/ijms23073980

Figure Lengend Snippet: Canonical or non-canonical STAT1 pathway protein expression. Cells were activated with IFN-γ or IFN-γ+TNF-α and treated with 2 mM butyrate (green bars) for 1 h, 4 h or 16 h. IRF9 ( A , E , F ), phosphorylated JAK2 ( B , G – I ), phosphorylated STAT1 ( C , J – L ) and phosphorylated NFkB p65 ( D , M – O ) protein expression was determined. Representable blots of proteins of interest and β-actin control are shown in A-D. Data are represented as mean ± SEM ( n = 3). Significant differences are shown as * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.001, medium control as compared to 2 mM butyrate control or to IFN-γ or IFN-γ+TNF-α-activated intestinal epithelial cells in absence of butyrate. Activated cells were also compared to activated cells treated with butyrate.

Article Snippet: As primary antibodies phosphorylated JAK2 (1:200, Thermo Fisher Scientific), phosphorylated STAT1 (1:200, R&D systems), phosphorylated NF-kappaB p65 (1:1000, Cell signaling, Danvers, MA, USA), IRF9 (1:1000, Cell signaling) and β-actin (1:1000, Cell signaling) were used.

Techniques: Expressing, Control

Journal: International Journal of Molecular Sciences

Article Title: Butyrate Prevents Induction of CXCL10 and Non-Canonical IRF9 Expression by Activated Human Intestinal Epithelial Cells via HDAC Inhibition

doi: 10.3390/ijms23073980

Figure Lengend Snippet: The effect of butyrate and histone deacetylase (HDAC) inhibitor Trichostatin A (TSA) on mRNA expression of genes related to the STAT1 signaling cascade. Intestinal epithelial cells were activated with IFN-γ or IFN-γ+TNF-α and treated with 2 mM butyrate (green bars) or 10 μM TSA (pink bars) for 4 h. mRNA expression was measured in CXCL10 ( A , B ), IRF9 ( C , D ) JAK2 ( E , F ) and SOCS1 ( G , H ). Data are represented as mean ± SEM ( n = 4). Significant differences are shown as ** p < 0.01, *** p < 0.001, **** p < 0.001 Control compared to butyrate, TSA, IFN-γ ( A , C , E , G ) or IFNγ+TNFα ( B , D , F , H ) activated cells and activated cells compared to butyrate or TSA treated activated cells.

Article Snippet: As primary antibodies phosphorylated JAK2 (1:200, Thermo Fisher Scientific), phosphorylated STAT1 (1:200, R&D systems), phosphorylated NF-kappaB p65 (1:1000, Cell signaling, Danvers, MA, USA), IRF9 (1:1000, Cell signaling) and β-actin (1:1000, Cell signaling) were used.

Techniques: Histone Deacetylase Assay, Expressing, Control

Journal: International Journal of Molecular Sciences

Article Title: Butyrate Prevents Induction of CXCL10 and Non-Canonical IRF9 Expression by Activated Human Intestinal Epithelial Cells via HDAC Inhibition

doi: 10.3390/ijms23073980

Figure Lengend Snippet: The effect of histone deacetylase inhibitor Trichostatin A (TSA) on downstream proteins of the STAT1 signaling cascade. Protein expression in IFN-γ or IFN-γ+TNF-α-activated intestinal epithelial cells (IECs) after 4 h incubation with 10 μM TSA (pink bars). Proteins measured were IRF9 ( A , E ), phosphorylated JAK2 ( B , F ), phosphorylated STAT1 ( C , G ), phosphorylated NFκB p65 ( D , H ). Data are represented as mean ± SEM ( n = 3). Significant differences are shown as * p < 0.05, ** p < 0.01, *** p < 0.001, control compared to IFN-γ-activated IECs, IFN-γ+TNF-α-activated IECs or 10 μM TSA control. Activated cells were compared to activated cells treated with TSA.

Article Snippet: As primary antibodies phosphorylated JAK2 (1:200, Thermo Fisher Scientific), phosphorylated STAT1 (1:200, R&D systems), phosphorylated NF-kappaB p65 (1:1000, Cell signaling, Danvers, MA, USA), IRF9 (1:1000, Cell signaling) and β-actin (1:1000, Cell signaling) were used.

Techniques: Histone Deacetylase Assay, Expressing, Incubation, Control

Journal: Frontiers in Immunology

Article Title: HSP90AA1 interacts with CSFV NS5A protein and regulates CSFV replication via the JAK/STAT and NF-κB signaling pathway

doi: 10.3389/fimmu.2022.1031868

Figure Lengend Snippet: HSP90AA1 over-expression activates STAT1 and P65 in PK-15 and 3D4/2 cells. (A) HSP90AA1 over-expression affected the phosphorylation of STAT1 and nuclear translocation of p-STAT1 in PK-15 and 3D4/2 cells were observed by laser confocal microscopy. (B) HSP90AA1 over-expression affected the nuclear translocation of p-P65 in PK-15 and 3D4/2 cells were observed by laser confocal microscopy.

Article Snippet: Primary antibodies include rabbit anti-p-STAT1 pAb (Bioss, bs-3427R), rabbit-anti-p-P65 pAb (Affinity Biosciences, AF2006) and mouse anti-Flag mAb.

Techniques: Over Expression, Translocation Assay, Confocal Microscopy

Journal: Frontiers in Immunology

Article Title: HSP90AA1 interacts with CSFV NS5A protein and regulates CSFV replication via the JAK/STAT and NF-κB signaling pathway

doi: 10.3389/fimmu.2022.1031868

Figure Lengend Snippet: CSFV infection antagonizes the activation of HSP90AA1 on JAK/STAT pathway. (A, B) Western blot for JAK1, STAT1, p-JAK1, p-STAT1, OAS2, ISG-15 and HSP90AA1 expression in HSP90AA1-overexpression or knock-downed PK-15 and 3D4/2 cells. Cells were infected with CSFV (MOI=1) after transfection p3×Flag-HSP90AA1 or siHSP90AA1. The cells were not infected with CSFV as a control. Cells were harvested at 24hpi and 48hpi served to Western blot. The relative levels of proteins were estimated by histograms representing density reading of the gel bands with Image J, and the ratios were calculated relative to tubulin control. (*p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 calculated using two-way ANOVA, ns, not significant).

Article Snippet: Primary antibodies include rabbit anti-p-STAT1 pAb (Bioss, bs-3427R), rabbit-anti-p-P65 pAb (Affinity Biosciences, AF2006) and mouse anti-Flag mAb.

Techniques: Infection, Activation Assay, Western Blot, Expressing, Over Expression, Transfection