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: Cell Communication and Signaling : CCS

Article Title: An inhibitory effect on the nuclear accumulation of phospho-STAT1 by its unphosphorylated form

doi: 10.1186/s12964-022-00841-3

Figure Lengend Snippet: A , B Cytoplasmic retention of Flag-tagged WT STAT1 in IFNγ-stimulated cells co-expressing R602L/Y701F-GFP. Transfected STAT1-negative U3A and HeLa cells expressing R602L/Y701F-GFP and WT-Flag were treated with 50 ng/ml IFNγ for 45 min and subsequently stained with an anti-Flag antibody. A The fluorescence micrographs show the intracellular distribution of Flag-tagged STAT1 co-expressed in cells transfected with R602L/Y701F-GFP as well as the localization of the corresponding Hoechst-stained nuclei (n = 3 independent transfections). B The histogram demonstrates the nucleocytoplasmic STAT1-Flag distribution in the absence and presence of R602L/Y701F-GFP in IFNγ-treated HeLa cells, as determined by the ratio of nuclear-to-total fluorescence intensity (means ± standard deviations from n = 20 cells, * p ≤ 0.05, as assessed by a two-tailed Student’s t test). C The absence of nuclear P-STAT1 staining of the endogenous protein in HeLa cells expressing R602L/Y701F-GFP. HeLa cells were transfected with R602L/Y701F-GFP and stimulated with IFNγ for indicated times before staining with an anti-phospho-tyrosine antibody. D Crystal structure of the STAT1 anti-parallel dimer. A critical arginine residue at position 274 marked in cyan in the coiled-coil domains and threonine residue at position 385 marked in pink in the DNA-binding domains of the two STAT1 proteins (white and orange). Structural data were obtained from the Protein Data Bank (pdb) file 1YVL for the STAT1 anti-parallel dimer. E The absence of nuclear P-STAT1 staining of endogenous STAT1 in HeLa cells expressing R274W/R602L/Y701F-GFP. HeLa cells were transfected with R274W/R602L/Y701F-GFP and stimulated with IFNγ for indicated times before being stained with an anti-phospho-tyrosine antibody. Scale bars in A , C , E mark a distance of 10 µm

Article Snippet: The membranes were incubated with either a rabbit monoclonal phospho-Tyr701-specific STAT1 antibody (Cell Signaling Technology, 58D6) or monoclonal pan-STAT1 antibody (Cell Signaling Technology, D1K9Y) overnight.

Techniques: Expressing, Transfection, Staining, Fluorescence, Two Tailed Test, Residue, Binding Assay

Journal: Cell Communication and Signaling : CCS

Article Title: An inhibitory effect on the nuclear accumulation of phospho-STAT1 by its unphosphorylated form

doi: 10.1186/s12964-022-00841-3

Figure Lengend Snippet: The dimerization-deficient quadruple mutant (QM-GFP) mimicking U-STAT1 inhibits the detection of co-expressed WT protein but does not affect tyrosine phosphorylation. A – C HeLa cells were transfected with a plasmid coding for QM-GFP and stimulated with IFNγ for 45 min followed by staining with anti-phospho-tyrosine antibody. A The fluorescence micrographs show the intracellular distribution of QM-GFP and WT P-STAT1, as well as the localization of the corresponding Hoechst-stained nuclei. B Histogram demonstrating the net reduction of nuclear P-STAT1 intensity in HeLa cells expressing QM-GFP, as determined by the ratio of nuclear-to-total fluorescence intensity (n = 3 independent transfections, means ± standard deviations from n = 20 cells, * p ≤ 0.05 by a two-tailed Student’s t test). C Concentration-dependent inhibition of nuclear P-STAT1 accumulation by co-expressed QM-GFP. The fluorescence intensities of nuclear P-STAT1 staining were plotted against the total cellular QM-GFP fluorescence. D , E Representative immunoblot of whole cell extracts from STAT1-negative U3A cells co-expressing untagged STAT1 and QM-GFP after treatment for 45 min with 50 ng/ml of recombinant IFNγ and the quantification thereof from three independent transfection experiments. F , G An in vitro phosphorylation assay demonstrated no difference in tyrosine phosphorylation rates of the WT STAT1 by JAK2 with respect to the presence or absence of QM-GFP. Whole cell extracts (10 μl in each reaction) from reconstituted U3A cells expressing untagged STAT1 in combination with WT-GFP or QM-GFP were incubated with 4 μg/ml of recombinant JAK2 kinase and the levels of P-STAT1 were monitored over time by means of Western blotting (n = 3). Statistical analysis revealed no significant difference in the phosphorylation kinetics of the WT in the presence of either QM-GFP or WT-GFP. H , I Results from an in vitro dephosphorylation assay using extracts from IFNγ-pre-stimulated U3A cells expressing untagged STAT1 in combination with WT-GFP or QM-GFP (10 μl each) incubated for 0, 15 and 30 min with 2 U of the STAT-specific Tc45 phosphatase (n = 3). Tyrosine dephosphorylation was followed by immunoblotting including a quantitative analysis of the phospho-tyrosine signals divided by total amount of STAT1 signal. Scale bar in A marks a distance of 10 µm

Article Snippet: The membranes were incubated with either a rabbit monoclonal phospho-Tyr701-specific STAT1 antibody (Cell Signaling Technology, 58D6) or monoclonal pan-STAT1 antibody (Cell Signaling Technology, D1K9Y) overnight.

Techniques: Mutagenesis, Phospho-proteomics, Transfection, Plasmid Preparation, Staining, Fluorescence, Expressing, Two Tailed Test, Concentration Assay, Inhibition, Western Blot, Recombinant, In Vitro, Incubation, De-Phosphorylation Assay

Journal: Cell Communication and Signaling : CCS

Article Title: An inhibitory effect on the nuclear accumulation of phospho-STAT1 by its unphosphorylated form

doi: 10.1186/s12964-022-00841-3

Figure Lengend Snippet: Unaltered DNA binding, reporter activation and target gene induction by WT STAT1 in the presence of a dimerization-deficient mutant. A Electrophoretic mobility shift assay demonstrated unchanged binding of the recombinant WT protein from reconstituted U3A cell extracts to a [ 33 P]-radioactively labelled M67 probe containing a single GAS sequence, whereas the co-expressed unphosphorylated STAT1 variants and empty vector (pEGFPN1) showed no DNA-binding indicated by the absence of a GFP-tagged STAT1 band in lanes 3 to 5. The asterisk at the margin of the gelshift indicates an unspecific band. B EMSA result showing the unaltered capacity of endogenous STAT1 from HeLa cell extracts to form tetramers on the radioactively-labelled DNA probe 2xGAS, containing two GAS sites in tandem orientation, in the presence of co-expressed QM-GFP. HeLa cells were either untransfected (UT) or transfected with the indicated expression plasmids, and on the next day the cells were treated for 45 min with IFNγ or left untreated. Extracts were incubated with 2xGAS DNA probed for 15 min before subsequently being challenged by a 750-fold molar excess of unlabelled M67 DNA incubated for 10 min at RT in a competition assay (comp). For super-shift assays, 20 ng of STAT1- (αS1) or STAT3-specific (αS3) antibody were added to the reaction for 40 min at RT. C , D EMSA result showing the unaltered cytoplasmic and nuclear fractions of endogenous P-STAT1 from HeLa cell lysates bound to a [ 33 P]-radioactively labelled M67 probe containing a single GAS sequence, as the additional presence of QM-GFP did not hamper nuclear import of the endogenous STAT1 but its nuclear retention. E HeLa cells expressing WT-GFP, empty vector (pEGFPN1) or the quadruple mutant QM-GFP were left untreated (−) or stimulated for 6 h with 50 ng/ml of IFNγ (+). In whole extracts from these cells, luciferase luminescence of a reporter construct with a triple GAS site (3xLy6E) and the enzymatic activity of the co-expressed β-galactosidase were measured and represented graphically. F U3A cells were transfected with a STAT1-responsive luciferase reporter construct, a β-galactosidase expression vector and a combination of equal amounts of WT-GFP and the indicated STAT1 variants. These cells were either unstimulated (−) or stimulated with 50 ng/ml of IFNγ (+) and subsequently luciferase activity, normalized to the β-galactosidase expression, was measured in whole cell extracts and represented graphically. The experiment was repeated in six independent transfections at least three times. G – J U3A cells were untransfected (UT) or transfected with a plasmid coding for WT-GFP alone or a combination of WT-GFP and the indicated U-STAT1 variants or empty GFP vector (pEGFPN1). These cells where either untreated or stimulated with 50 ng/ml of IFNγ for 6 h. after which RNA was isolated and converted to cDNA. Histograms show the results from qPCR experiments for the following STAT1 target genes: G GBP1 , H MIG , I CXCL10 and J IRF1 . Gene induction was normalized to the expression of the house-keeping gene GAPDH . Histograms show means and standard deviations wherein the significant differences between the IFNγ-stimulated variant samples and cells expressing a single transfection of WT protein are marked by bars and asterisks. The experiment was repeated three times

Article Snippet: The membranes were incubated with either a rabbit monoclonal phospho-Tyr701-specific STAT1 antibody (Cell Signaling Technology, 58D6) or monoclonal pan-STAT1 antibody (Cell Signaling Technology, D1K9Y) overnight.

Techniques: Binding Assay, Activation Assay, Mutagenesis, Electrophoretic Mobility Shift Assay, Recombinant, Sequencing, Plasmid Preparation, Transfection, Expressing, Incubation, Competitive Binding Assay, Luciferase, Construct, Activity Assay, Isolation, Variant Assay

Journal: Cell Communication and Signaling : CCS

Article Title: An inhibitory effect on the nuclear accumulation of phospho-STAT1 by its unphosphorylated form

doi: 10.1186/s12964-022-00841-3

Figure Lengend Snippet: Deletion of the amino-terminus in mutant U-STAT1 restores nuclear accumulation of co-expressed, endogenous P-STAT1. A , B HeLa cells were transfected with an expression plasmid coding for R602L/Y701F-GFP, QM-GFP, ΔN/R602L/Y701F-GFP and QM/L407A/L409A-GFP and stimulated for 0 min or 45 min with 50 ng/ml of recombinant IFNγ, followed by staining with an anti-phospho-tyrosine antibody. Fluorescence micrographs show restored nuclear accumulation of P-STAT1 in the presence of amino-terminal deletion mutant ΔN/R602L/Y701F-GFP and the dsNLS mutant QM/L407A/L409A-GFP. Histograms show the quantification of nuclear P-STAT1 from HeLa cells co-expressing the indicated STAT1 variants in comparison with untransfected HeLa cells from three independent experiments (n = 3, means ± standard deviations from n = 20 cells, * p ≤ 0.05). C Fluorescence micrographs displaying the absence of nuclear endogenous P-STAT1 staining in the presence of mutant U-STAT1 constructs expressed in HeLa cells stimulated for 0 min and 45 min with recombinant IFNα (50 ng/ml). Note the rescue effect mediated by the deletion of the amino-terminus in the construct ΔN/R602L/Y701F-GFP under stimulation with type I IFN. Scale bars in A , C mark a distance of 10 µm. D Cellular fractionation experiments using cytoplasmic and nuclear lysates from HeLa cells expressing the indicated STAT1 variants demonstrate the presence of endogenous phospho-STAT1 in both compartments in IFNγ-pretreated cells. Representative immunoblots demonstrate unchanged tyrosine phosphorylation of endogenous STAT3 in whole cell extracts from HeLa cells expressing U-STAT1, after stimulation with IFNγ (lower panels). E , F Quantification from immunoblots of whole extracts from HeLa cells expressing the indicated mutants, including the quadruple mutant (QM), before and after treatment for 45 min with 50 ng/ml of recombinant IFNγ from three independent transfection experiments for the expression of phospho-STAT1 ( E ) and the expression of phospho-STAT3 ( F )

Article Snippet: The membranes were incubated with either a rabbit monoclonal phospho-Tyr701-specific STAT1 antibody (Cell Signaling Technology, 58D6) or monoclonal pan-STAT1 antibody (Cell Signaling Technology, D1K9Y) overnight.

Techniques: Mutagenesis, Transfection, Expressing, Plasmid Preparation, Recombinant, Staining, Fluorescence, Comparison, Construct, Cell Fractionation, Western Blot, Phospho-proteomics

Journal: Cell Communication and Signaling : CCS

Article Title: An inhibitory effect on the nuclear accumulation of phospho-STAT1 by its unphosphorylated form

doi: 10.1186/s12964-022-00841-3

Figure Lengend Snippet: Proposed model of U-STAT1-dependent import modulation of P-STAT1. The left panel describes a condition of low-level cellular U-STAT1, wherein fully functional import complexes of P-STAT1 are formed and translocated to the nucleus in cytokine-stimulated cells. The right panel depicts a high concentration of U-STAT1, wherein U-STAT1 interacts through its amino-terminus and dsNLS with both components of the importin dimer. The formation for a full-fledged import complex is hindered by the missing tyrosine phosphorylation residue of U-STAT1

Article Snippet: The membranes were incubated with either a rabbit monoclonal phospho-Tyr701-specific STAT1 antibody (Cell Signaling Technology, 58D6) or monoclonal pan-STAT1 antibody (Cell Signaling Technology, D1K9Y) overnight.

Techniques: Functional Assay, Concentration Assay, Phospho-proteomics, Residue

Journal: PLoS ONE

Article Title: SLFN11 can sensitize tumor cells towards IFN-γ-mediated T cell killing

doi: 10.1371/journal.pone.0212053

Figure Lengend Snippet: A) Schematic overview of experimental design in B. B) Growth kinetics of cells left untreated or treated with 10 ng/ml of IFN-γ for 24 h, in the presence or absence of Q-VD-OPh. C). Western blot analysis of phosphorylated STAT1 in HAP1 cells transduced with a control lentiviral vector or with lentiviral vectors encoding independent SLFN11-targeting shRNA, either left untreated or exposed to 10 ng/ml of IFN-γ for 24h. D) IRF1 transcript levels following exposure to 10 ng/ml of IFN-γ for the indicated times in parental, IFNGR1 KO, and SLFN11 KO cells.

Article Snippet: The following antibodies were used for western blot analysis: anti-HSP90: H114 (SantaCruz); anti-TUBA1A: 2144s (Cell Signaling Technology); anti-phosphorylated STAT1 7649s (Cell Signaling Technology); anti γ-H2AX 2577s (Cell Signaling Technology); anti-phosphorylated CHK1 12302s (Cell Signaling Technology); anti-phosphorylated ATM 10H11.E12 (Millipore); anti-SLFN11 HPA023030 (Atlas).

Techniques: Western Blot, Transduction, Control, Plasmid Preparation, shRNA

Journal: Journal of Biological Chemistry

Article Title: Inhibitor of κB Kinase ϵ (IKKϵ), STAT1, and IFIT2 Proteins Define Novel Innate Immune Effector Pathway against West Nile Virus Infection*

doi: 10.1074/jbc.m111.285205

Figure Lengend Snippet: FIGURE 2. Virus infection induces delayed STAT1 Ser-708 phosphorylation. A, WT or IKK/ MEFs were mock-stimulated or stimulated with 100IU/ml IFN-. Protein lysate was collected 16 h post-IFN stimulation and immunoblotted using p-STAT1 Ser-708, p-STAT1 Ser-727, p-STAT1 Tyr-701, and total STAT1 antibodies. B and C, Sendai and WNV virus infections induce STAT1 Ser-708 phosphorylation. HEK293 cells were infected with 100 HA units/ml SenV (B) or West Nile virus strain Madagascar (WNV-MAD) (C) at an m.o.i. of 1. At the indicated times following infection, protein lysates were collected and immunoblotted for p-STAT1 Ser-708, p-STAT1 Tyr-701, total STAT1, p-IRF-3, total IRF-3, IFIT1, and SenV or WNV. Tubulin and GAPDH were used as loading controls.

Article Snippet: The following primary antibodies were used for immunoblot analyses: -ADAR1 (Abnova); -IRF-3 (M. David, UCSD); -IFIT1, -murine IFIT2 and -murine IFIT3 (G. Sen, Cleveland Clinic); -ISG15 (A. Haas, Louisiana State University); -WNV (Centers for Disease Control and Prevention); -pSTAT1 Tyr-701, -p-STAT1 Ser-727, -STAT1, -p-IRF-3 (Cell Signaling Technology, Inc.); -murine IRF-3 (Invitrogen); -IKK (Imgenex); -PKR (Santa Cruz Biotechnology, Inc.); -SenV (Biodesign International); -FLAG (M2), and -Tubulin (Sigma).

Techniques: Virus, Infection, Phospho-proteomics

Journal: Journal of Biological Chemistry

Article Title: Inhibitor of κB Kinase ϵ (IKKϵ), STAT1, and IFIT2 Proteins Define Novel Innate Immune Effector Pathway against West Nile Virus Infection*

doi: 10.1074/jbc.m111.285205

Figure Lengend Snippet: FIGURE3.TypeI,typeII,andtypeIIIIFNsinduceSTAT1Ser-708phosphor- ylation. 2fTGH cells were mock-treated or treated with 100 IU/ml IFN- (A) or 50 ng/ml IFN- (B). C, PH5CH8 cells were mock-treated (lane 1), treated with 100 ng/ml IFN-1 (lanes 2-6), or 100 IU/ml IFN- (lanes 7-9). Protein lysate was collected at respective time points following IFN treatment and immuno- blotted for p-STAT1 Ser-708, p-STAT1 Tyr-701, p-STAT1 Ser-727, total STAT1, ADAR1, and IFIT1.

Article Snippet: The following primary antibodies were used for immunoblot analyses: -ADAR1 (Abnova); -IRF-3 (M. David, UCSD); -IFIT1, -murine IFIT2 and -murine IFIT3 (G. Sen, Cleveland Clinic); -ISG15 (A. Haas, Louisiana State University); -WNV (Centers for Disease Control and Prevention); -pSTAT1 Tyr-701, -p-STAT1 Ser-727, -STAT1, -p-IRF-3 (Cell Signaling Technology, Inc.); -murine IRF-3 (Invitrogen); -IKK (Imgenex); -PKR (Santa Cruz Biotechnology, Inc.); -SenV (Biodesign International); -FLAG (M2), and -Tubulin (Sigma).

Techniques:

Journal: Journal of Biological Chemistry

Article Title: Inhibitor of κB Kinase ϵ (IKKϵ), STAT1, and IFIT2 Proteins Define Novel Innate Immune Effector Pathway against West Nile Virus Infection*

doi: 10.1074/jbc.m111.285205

Figure Lengend Snippet: FIGURE 4. Signaling through IFNAR is required for STAT1 Ser-708 phosphorylation following type I IFN treatment or virus infection. WT, IRF-3/, or IFNAR/ (A) and parental 2fTGH cells or their derivative U5A cells (which lack IFNAR) (B) were infected with WNV-MAD at an m.o.i. of 1. Protein lysates were collected at the indicated time points and immunoblotted for p-STAT1 Ser-708, p-STAT1 Tyr-701, p-STAT1 Ser-727, total STAT1, p-IRF-3, total IRF-3, and WNV. C and D, the same cells were also mock-stimulated or stimulated with 100 IU/ml IFN- for 6 or 16 h. Protein lysates were collected at the indicated time points and immunoblotted for p-STAT1 Ser-708, p-STAT1 Tyr-701, p-STAT1 Ser-727, total STAT1, p-IRF-3, total IRF-3, IFIT2, IFIT3, and IFIT1. Asterisk, nonspecific band.

Article Snippet: The following primary antibodies were used for immunoblot analyses: -ADAR1 (Abnova); -IRF-3 (M. David, UCSD); -IFIT1, -murine IFIT2 and -murine IFIT3 (G. Sen, Cleveland Clinic); -ISG15 (A. Haas, Louisiana State University); -WNV (Centers for Disease Control and Prevention); -pSTAT1 Tyr-701, -p-STAT1 Ser-727, -STAT1, -p-IRF-3 (Cell Signaling Technology, Inc.); -murine IRF-3 (Invitrogen); -IKK (Imgenex); -PKR (Santa Cruz Biotechnology, Inc.); -SenV (Biodesign International); -FLAG (M2), and -Tubulin (Sigma).

Techniques: Phospho-proteomics, Virus, Infection

Journal: Journal of Biological Chemistry

Article Title: Inhibitor of κB Kinase ϵ (IKKϵ), STAT1, and IFIT2 Proteins Define Novel Innate Immune Effector Pathway against West Nile Virus Infection*

doi: 10.1074/jbc.m111.285205

Figure Lengend Snippet: FIGURE 5. STAT1 Ser-708 phosphorylation requires de novo protein syn- thesis, STAT1 tyrosine dephosphorylation, and nuclear export. A, 2fTGH cells were mock-treated (-CHX, lanes 1-5) or treated with CHX (CHX, lanes 6-11) to block protein synthesis. At 30 min (lanes 6-10) or 16 h (lane 11) follow- ing CHX treatment, cells were mock-stimulated (M) or stimulated with IFN-. Cells were harvested at 10 min as well as 1, 6, and 16 h post-IFN stimulation andimmunoblottedtodetectp-STAT1Ser-708,p-STAT1Tyr-701,totalSTAT1, ISG15, and IFIT1. B, 2fTGH cells were not treated (NT, lanes 1-3), pretreated with 100 nM LMB (lanes 4-6), or 50 mM pervanadate (Van, lanes 7-9) 1 h before mock stimulation or stimulation with 100 IU/ml IFN-. Cells were harvested at 1 and 16 h post-stimulation. Immunoblot analysis was performed using p-STAT1 Ser-708, p-STAT1 Tyr-701, p-STAT1 Ser-727, total STAT1, ADAR1, and IFIT1 antibodies.

Article Snippet: The following primary antibodies were used for immunoblot analyses: -ADAR1 (Abnova); -IRF-3 (M. David, UCSD); -IFIT1, -murine IFIT2 and -murine IFIT3 (G. Sen, Cleveland Clinic); -ISG15 (A. Haas, Louisiana State University); -WNV (Centers for Disease Control and Prevention); -pSTAT1 Tyr-701, -p-STAT1 Ser-727, -STAT1, -p-IRF-3 (Cell Signaling Technology, Inc.); -murine IRF-3 (Invitrogen); -IKK (Imgenex); -PKR (Santa Cruz Biotechnology, Inc.); -SenV (Biodesign International); -FLAG (M2), and -Tubulin (Sigma).

Techniques: Phospho-proteomics, De-Phosphorylation Assay, Blocking Assay, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Inhibitor of κB Kinase ϵ (IKKϵ), STAT1, and IFIT2 Proteins Define Novel Innate Immune Effector Pathway against West Nile Virus Infection*

doi: 10.1074/jbc.m111.285205

Figure Lengend Snippet: FIGURE 6. IKK mediates IFIT2 expression and protection against WNV pathogenesis in vivo. WT Bl6 and IKK/ mice were mock-infected (PBS only) or infected with 103 pfu of WNV-MAD subcutaneously through footpad injection. A, mice were monitored and scored daily for clinical symptoms over 17 days. Clinical scores from four representative mice per group were graphed. B, spleens from WT or IKK/ mice, mock-infected or infected with WNV-MAD, were collected at days 4, 6, and 12 post-infection. Protein lysates were extracted by homogenizing spleens with radioimmune precipitation assay buffer and immunoblotted using p-STAT1 Ser-708, p-STAT1 Tyr-701, p-STAT1 Ser-727, total STAT1, IFIT2, IFIT1, WNV, and IKK antibodies. The immunoblot analysis panel is a representative from four mice per infection group.

Article Snippet: The following primary antibodies were used for immunoblot analyses: -ADAR1 (Abnova); -IRF-3 (M. David, UCSD); -IFIT1, -murine IFIT2 and -murine IFIT3 (G. Sen, Cleveland Clinic); -ISG15 (A. Haas, Louisiana State University); -WNV (Centers for Disease Control and Prevention); -pSTAT1 Tyr-701, -p-STAT1 Ser-727, -STAT1, -p-IRF-3 (Cell Signaling Technology, Inc.); -murine IRF-3 (Invitrogen); -IKK (Imgenex); -PKR (Santa Cruz Biotechnology, Inc.); -SenV (Biodesign International); -FLAG (M2), and -Tubulin (Sigma).

Techniques: Expressing, In Vivo, Infection, Injection, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Inhibitor of κB Kinase ϵ (IKKϵ), STAT1, and IFIT2 Proteins Define Novel Innate Immune Effector Pathway against West Nile Virus Infection*

doi: 10.1074/jbc.m111.285205

Figure Lengend Snippet: FIGURE 7. A model illustrating that early and late ISGs induction is regulated by multiple STAT1 posttranslational modifications. 1, the canonical JAK-STAT signaling is activated following type I IFN binding to its receptor, which results in STAT1 Tyr-701 phosphorylation, ISGF3 formation, and its nuclear translocation. ISGF3 binding to the ISRE element induces transcription of ISGs. 2, chromatin-bound STAT1 can be phosphorylated by MAPK at residue Ser-727, whichinducesitssumoylation.3and4,nuclearSTAT1isalsoacetylatedbyhistoneacetyltransferase(HAT)CREB-bindingprotein(CBP),resultinginrecruitment of TCP1, which catalyzes STAT1 tyrosine dephosphorylation. Sumoylated-acetylated STAT1 cycles back to the cytoplasm, and both modifications render STAT1 unable to be further tyrosine-phosphorylated. 5 and 6, type I IFN signaling and unknown IFN-stimulated factor(s) activate IKK phosphorylation of STAT1 Ser-708. 7, STAT1 molecules phosphorylated at Ser-708 can enter the nucleus and induce expression of a specific ISG subset. pY, tyrosine phosphorylation; pS, serine phosphorylation; Ac, acetylation; Su, sumoylation.

Article Snippet: The following primary antibodies were used for immunoblot analyses: -ADAR1 (Abnova); -IRF-3 (M. David, UCSD); -IFIT1, -murine IFIT2 and -murine IFIT3 (G. Sen, Cleveland Clinic); -ISG15 (A. Haas, Louisiana State University); -WNV (Centers for Disease Control and Prevention); -pSTAT1 Tyr-701, -p-STAT1 Ser-727, -STAT1, -p-IRF-3 (Cell Signaling Technology, Inc.); -murine IRF-3 (Invitrogen); -IKK (Imgenex); -PKR (Santa Cruz Biotechnology, Inc.); -SenV (Biodesign International); -FLAG (M2), and -Tubulin (Sigma).

Techniques: Binding Assay, Phospho-proteomics, Translocation Assay, Residue, De-Phosphorylation Assay, Expressing

Journal: Molecular pharmacology

Article Title: Synergy between Interleukin-1 β , Interferon- γ , and Glucocorticoids to Induce TLR2 Expression Involves NF- κ B, STAT1, and the Glucocorticoid Receptor.

doi: 10.1124/molpharm.123.000740

Figure Lengend Snippet: Fig. 5. Effect of IL-1b, glucocorticoid, and IFN-c on STAT1 phosphorylation. A549 cells were either untreated or treated with IFN-c (10 ng/ml) prior to harvesting for western blot analysis at the indicated times. Representative blots of (A) phospho-STAT1 (P-STAT1 Y701) and GAPDH and (B) P-STAT1 (S727), total STAT1, and GAPDH from N independent experiments are shown. In each case, densitometric data normalized to GAPDH are plotted as means ± S.D. (error bars). Significance relative to untreated at 15 minutes was assessed by ANOVA with a Dunnett’s mul- tiple comparison test. (C) A549 cells were either not treated or treated with IL-1b (1 ng/ml), IFN-c (10 ng/ml), and/or Dex (1 mM) as indicated. Cells were harvested at 1 hour for western blot analysis of P-STAT1 (Y701 and S727) and GAPDH. Representative blots of P-STAT1 (Y701 & S727) and GAPDH from N independent experiments are shown. Densitometric data for P-STAT1 (Y701) (upper graph) and P-STAT1 (S727) (lower graph) were normalized to GAPDH, expressed as means ± S.D., and plotted as bar graphs overlaid with scatter plots. Significance relative to un- treated (*) or IL-1b1IFN-c (#) was tested using ANOVA with a Tukey post hoc test. *, #P # 0.05; **, ##P # 0.01; ***, ###P # 0.001. Scatterplots for all data in (A) and (B) are shown as Supplemental Fig. 6.

Article Snippet: After blocking, membranes were probed with primary antibodies against TLR2 (12276, Cell Signaling; RRID: AB_2797867 and ab108998, Abcam; RRID: AB_10861644), IjBa (sc371, SantaCruz; RRID: AB_10861644), Ser32/Ser36 phosphorylated IjBa (9246, Cell Signaling; RRID: AB_2267145), p65 (sc-8008, SantaCruz RRID: AB_628017), Ser536 phosphorylated p65 (3036, Cell Signaling; RRID: AB_331281), GR (PA1-511A, Thermo Fisher Scientific; RRID: AB_2236340), Tyr701 phosphorylated STAT1 (9167, Cell Signaling; RRID: AB_561284), Ser727 phosphorylated STAT1 (9177, Cell Signaling; RRID: AB_2197983), STAT1 (9172, Cell Signaling; RRID: AB_2198300), or GAPDH (MCA4739, Bio-Rad; RRID: AB_1720065).

Techniques: Phospho-proteomics, Western Blot, Comparison

Journal: Molecular pharmacology

Article Title: Synergy between Interleukin-1 β , Interferon- γ , and Glucocorticoids to Induce TLR2 Expression Involves NF- κ B, STAT1, and the Glucocorticoid Receptor.

doi: 10.1124/molpharm.123.000740

Figure Lengend Snippet: Fig. 6. Effect of JAK inhibitors on STAT1 phosphorylation. (A) A549 cells were pretreated with AZD1480, barcitinib and ruxolitinib at the indi- cated concentrations for 30 minutes, followed by stimulation with IFN-c (10 ng/ml) for 1 hour. Cells were harvested for western blot analysis of phospho-STAT1 (P-STAT1) and GAPDH. Representative blots for P-STAT1 (Y701), P-STAT1 (S727), and GAPDH from five independent experi- ments are shown. (B) Densitometric data for Y701 P-STAT1 and S727 P-STAT1 from western blots shown in (A) were normalized to GAPDH and plotted as mean ± S.D. (C) Data were expressed as percent IFN-c effect for each of AZD1480, barcitinib, and ruxolitinib and plotted as means ± S.D. Log EC50 of each compound for the inhibition of P-STAT1 (Y701) and P-STAT1 (S727) is expressed as mean ± S.D. and shown as scatter plots in the inserts within each graph of (C). Note: one dataset was excluded for ruxolitinib, and the full data are shown as Supplemental Fig. 8. (D and E) A549 cells were either not treated or pretreated with AZD1480, barcitinib, and ruxolitinib, each at 1 mM, for 1 hour prior to stimulation with IL-1b (1 ng/ml) and IFN-c (10 ng/ml) as indicated. Cells were harvested at 1 hour for western blot analysis of P-STAT1 and GAPDH. Representative blots for P-STAT1 (Y701 & S727) and GAPDH from N independent experiments are shown. Densitometric data for (D) P-STAT (S727) and (E) P-STAT1 (Y701) were normalized to GAPDH, expressed as means ± S.D., and plotted as bar graphs overlaid with scatter plots. Significance relative to untreated (*), IFN-c (#), or IL-1b 1 IFN-c ($) was tested using ANOVA with a Tukey post hoc test. *, #, $P # 0.05; **, ##, $$P # 0.01; ***, ###, $$$P # 0.001.

Article Snippet: After blocking, membranes were probed with primary antibodies against TLR2 (12276, Cell Signaling; RRID: AB_2797867 and ab108998, Abcam; RRID: AB_10861644), IjBa (sc371, SantaCruz; RRID: AB_10861644), Ser32/Ser36 phosphorylated IjBa (9246, Cell Signaling; RRID: AB_2267145), p65 (sc-8008, SantaCruz RRID: AB_628017), Ser536 phosphorylated p65 (3036, Cell Signaling; RRID: AB_331281), GR (PA1-511A, Thermo Fisher Scientific; RRID: AB_2236340), Tyr701 phosphorylated STAT1 (9167, Cell Signaling; RRID: AB_561284), Ser727 phosphorylated STAT1 (9177, Cell Signaling; RRID: AB_2197983), STAT1 (9172, Cell Signaling; RRID: AB_2198300), or GAPDH (MCA4739, Bio-Rad; RRID: AB_1720065).

Techniques: Phospho-proteomics, Western Blot, Inhibition

Journal: Molecular pharmacology

Article Title: Synergy between Interleukin-1 β , Interferon- γ , and Glucocorticoids to Induce TLR2 Expression Involves NF- κ B, STAT1, and the Glucocorticoid Receptor.

doi: 10.1124/molpharm.123.000740

Figure Lengend Snippet: Fig. 7. Characterization of a STAT1-driven luciferase reporter. (A) A549 cells stably transfected with a luciferase reporter containing three copies of a consensus STAT1-binding motif [shown in (B)] were either not stimulated (NS) or stimulated with the indicated concentrations of IFN-c prior to harvesting for luciferase assay after 6 hours. Luciferase activity from N independent experiments was expressed as fold of NS and plotted as mean ± S.D. (B) (Upper panel) reporter construct showing three copies of the consensus/wild-type (WT; bold blue) or mutant (Mut; bold blue 1 red) STAT1-binding motif is shown. KpnI and XhoI restriction sites used to clone the construct in pGL3.TATA.neo vector are underlined, with cut sites shown as black arrowheads. (Lower panel) A549 cells were stably transfected with the WT or Mut STAT1 reporter constructs and either NS or treated with IFN-c (10 ng/ml) for 6 hours followed by harvesting for luciferase assay. Luciferase activity from N independent experiments was expressed as fold of NS, and mean ± S.D. was plotted as bar graphs overlaid with scatter plots. Significance relative to untreated cells was deter- mined using paired t test. (C) A549 cells were either NS or incubated with lipid control (lip) alone or with the indicated concentrations of either control siRNA pool (C si-pool), or STAT1 siRNA pool (STAT1 si-pool) for 48 hours. Cells were then incubated overnight with serum-free media prior to harvesting for western blot analysis. Representative blots of STAT1 and GAPDH from N independent experiments are shown. Data ob- tained from densitometry analysis were normalized to GAPDH, and mean ± S.D. was plotted as bar graphs overlaid with scatter plots. Signifi- cance relative to C si-pool at each siRNA concentration was tested by paired t test. *P # 0.05; **P # 0.01; ***P # 0.001. (D) A549 cells stably transfected with the WT STAT1 reporter construct were treated with lipid or siRNAs as described in (C) prior to stimulation with IFN-c (10 ng/ml) for 6 hours. Cells were harvested for luciferase assay, and raw luciferase units (RLU) from N independent experiments were expressed as mean ± S.D. and plotted as bar graphs overlaid with scatter plots. Significance relative to C si-pool at each siRNA concentration was tested by paired t test. (E) A549 cells stably transfected with the WT STAT1 reporter construct were either NS or pretreated with the indicated concentra- tions of AZD1480, barcitinib, or ruxolitinib for 30 minutes followed by stimulation with IFN-c (10 ng/ml). Cells were harvested for luciferase assay after 6 hours. Luciferase activity from N independent experiments was expressed as fold of NS and plotted as mean ± S.D. Scatterplots for all data in (A) and e are shown as Supplemental Fig. 9.

Article Snippet: After blocking, membranes were probed with primary antibodies against TLR2 (12276, Cell Signaling; RRID: AB_2797867 and ab108998, Abcam; RRID: AB_10861644), IjBa (sc371, SantaCruz; RRID: AB_10861644), Ser32/Ser36 phosphorylated IjBa (9246, Cell Signaling; RRID: AB_2267145), p65 (sc-8008, SantaCruz RRID: AB_628017), Ser536 phosphorylated p65 (3036, Cell Signaling; RRID: AB_331281), GR (PA1-511A, Thermo Fisher Scientific; RRID: AB_2236340), Tyr701 phosphorylated STAT1 (9167, Cell Signaling; RRID: AB_561284), Ser727 phosphorylated STAT1 (9177, Cell Signaling; RRID: AB_2197983), STAT1 (9172, Cell Signaling; RRID: AB_2198300), or GAPDH (MCA4739, Bio-Rad; RRID: AB_1720065).

Techniques: Luciferase, Stable Transfection, Transfection, Binding Assay, Activity Assay, Construct, Mutagenesis, Plasmid Preparation, Incubation, Control, Western Blot, Concentration Assay

Journal: Molecular pharmacology

Article Title: Synergy between Interleukin-1 β , Interferon- γ , and Glucocorticoids to Induce TLR2 Expression Involves NF- κ B, STAT1, and the Glucocorticoid Receptor.

doi: 10.1124/molpharm.123.000740

Figure Lengend Snippet: Fig. 9. Effect of p65, GR, and STAT1 knockdown on IL-1b, IFN-c, and dexamethasone-induced TLR2 expression in A549 cells. (A) A549 cells were incubated with pools of either control siRNAs (C si-pool), p65 siRNAs (p65 si-pool), GR siRNAs (GR si-pool), or STAT1 siRNAs (STAT1 si-pool) (1 nM each) for 48 hours. Cells were then incubated overnight with serum-free media prior to harvesting for western blot analysis. Representative blots of p65, GR, STAT1, and GAPDH from five independent experiments are shown. (B–E) A549 cells were incubated with siRNA pools as in (A), followed by treatment with IL-1b (1 ng/ml), IFN-c (10 ng/ml), and/or Dex (1 mM), as indicated, for 24 hours. Cells were harvested for western blot analysis. Representative blots of TLR2 and GAPDH from N independent experiments are shown. Densitometric data were normalized to GAPDH, expressed as fold of untreated, and mean ± S.D. were plotted as bar graphs overlaid with scatter plots. Significance relative to C si-pool in each case was tested using ANOVA with a Dunnett’s post hoc test. *P # 0.05; **P # 0.01; ***P # 0.001.

Article Snippet: After blocking, membranes were probed with primary antibodies against TLR2 (12276, Cell Signaling; RRID: AB_2797867 and ab108998, Abcam; RRID: AB_10861644), IjBa (sc371, SantaCruz; RRID: AB_10861644), Ser32/Ser36 phosphorylated IjBa (9246, Cell Signaling; RRID: AB_2267145), p65 (sc-8008, SantaCruz RRID: AB_628017), Ser536 phosphorylated p65 (3036, Cell Signaling; RRID: AB_331281), GR (PA1-511A, Thermo Fisher Scientific; RRID: AB_2236340), Tyr701 phosphorylated STAT1 (9167, Cell Signaling; RRID: AB_561284), Ser727 phosphorylated STAT1 (9177, Cell Signaling; RRID: AB_2197983), STAT1 (9172, Cell Signaling; RRID: AB_2198300), or GAPDH (MCA4739, Bio-Rad; RRID: AB_1720065).

Techniques: Knockdown, Expressing, Incubation, Control, Western Blot

Journal: The Journal of Immunology Author Choice

Article Title: Direct Antiviral Activity of IFN-Stimulated Genes Is Responsible for Resistance to Paramyxoviruses in ISG15-Deficient Cells

doi: 10.4049/jimmunol.1901472

Figure Lengend Snippet: Functional characterization of A549-ISG15–knockout cell lines. ( A ) CRISPR/Cas9 genome editing was used to knockout ISG15 expression in A549 cells, followed by single-cell cloning [following previously reported procedures ]. Four independent clones were treated with 1000 IU/ml IFN-α for 24 and 48 h or left untreated, and protein expression was tested by immunoblot analysis of ISG15, MxA, IFIT1, and β-actin. Control cells were naive A549 cells. Representative image from two independent experiments. ( B ) A549 and A549-ISG15 −/− (B8) cells were treated with 1000 IU/ml IFN-α for 30 min, then extensively washed and media without IFN replaced. Cells were harvested at 0 and 30 min and 24 h after IFN-α removal and phopho-STAT1, total STAT1, MxA, ISG15, and β-actin were detected following immunoblot analysis. ( C ) A549 and A549-ISG15 −/− (clone B8) were treated with 1000 IU/ml IFN-α for 24 h. Expression of ISGs was tested using reverse transcription quantitative PCR (RT-qPCR) with primers specific for HERC5 , USP18 , IFIT1 , and MxA . Relative expression was determined following SYBR Green quantitative PCR (qPCR) using ΔΔCt method. β-Actin expression was used to normalize between samples. Error bars represent the SD of the mean from three independent RNA samples. ( D ) A549 and A549-ISG15 −/− (clone B8) were treated with 1000 IU/ml for 24 h. Cells were washed, and fresh media (without IFN-α) were replaced. Cells were processed for immunoblot analysis using Abs specific for MxA and β-actin at 24 h post–IFN-α and every 24 h thereafter for 72 h. Controls were cells without IFN-α. Representative image from two independent experiments. ( E ) A549 and A5549-ISG15 −/− (clone B8) were treated with 1000 IU/ml for 24 and 48 h (or left untreated). Whole-cell lysates were analyzed by immunoblotting with Abs specific for USP18, ISG15, and β-actin. Image is representative of more than three independent experiments.

Article Snippet: Confluent monolayers in six-well dishes were lysed with 250 μl 2× Laemmli sample buffer (4% w/v SDS, 20% v/v glycerol, 0.004% w/v bromphenol blue, and 0.125 M Tris–HCl (pH 6.8) with 10% v/v 2-ME) for 10 min, incubated at 95°C for 10 min, sonicated at 4°C with three cycles of 30 s on/30 s off in a Bioruptor Pico (Diagenode) and clarified by centrifugation at 12,000 × g , 4°C for 10 min. SDS-PAGE in Tris-glycine-SDS running buffer and immunoblotting followed standard techniques using the following Abs: mouse monoclonal anti-ISG15 F-9 (catalog no. sc166755; Santa Cruz Biotechnology), rabbit polyclonal anti-MxA (catalog no. 13750-1-AP; Proteintech), goat polyclonal anti-IFIT1 N-16 (catalog no. sc82946; Santa Cruz Biotechnology), mouse monoclonal anti–β-ACTIN, UBA7 (anti-UBE1L B-7; catalog no. sc-390097; Santa Cruz Biotechnology), rabbit monoclonal anti–phosphorylated STAT1 (anti–phospho-STAT1 [Tyr 701 ] 58D6; catalog no. 9167; Cell Signaling Technology), mouse monoclonal anti-PIV5 NP 125 , mouse monoclonal anti-HPIV2 and anti-PIV5 phosphoprotein (P) 161 [Ab cross-reacts with P of both viruses ( )], and mouse monoclonal anti-HPIV3 NP ( ).

Techniques: Functional Assay, Knock-Out, CRISPR, Expressing, Cloning, Clone Assay, Western Blot, Control, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, SYBR Green Assay

Journal: The Journal of Immunology Author Choice

Article Title: Direct Antiviral Activity of IFN-Stimulated Genes Is Responsible for Resistance to Paramyxoviruses in ISG15-Deficient Cells

doi: 10.4049/jimmunol.1901472

Figure Lengend Snippet: IFN-α pretreatment of ISG15-deficient cells leads to virus resistance that is independent of ISGylation. ( A ) Control (naive A549) and four independent clones of A549-ISG15 −/− cells generated by CRISPR/Cas9 genome editing were treated with 1000 IU/ml IFN-α for 16 h or left untreated and then infected with PIV5-W3 (MOI = 10). Cells were harvested at 24 and 48 h p.i. and processed for immunoblot analysis using Abs specific for PIV5 NP, ISG15, STAT1, IFIT1, MxA, and β-actin. This experiment was independently performed twice. ( B ) UBA7-knockout cells were generated using CRISPR/Cas9 genome editing; Cas9-expressing A549 cells were first generated (following transduction with lentiCas9-Blast) and then transduced with lentiGuide-Puro expressing a sgRNA that targeted exon 3 of the UBA7 gene. Knockout cells were single-cell cloned, and three were selected for further analysis. These cells were treated with IFN-α or left untreated, infected, and processed as in (A) using Abs specific for PIV5 NP, ISG15, UBA7, and β-actin. This experiment was independently performed twice.

Article Snippet: Confluent monolayers in six-well dishes were lysed with 250 μl 2× Laemmli sample buffer (4% w/v SDS, 20% v/v glycerol, 0.004% w/v bromphenol blue, and 0.125 M Tris–HCl (pH 6.8) with 10% v/v 2-ME) for 10 min, incubated at 95°C for 10 min, sonicated at 4°C with three cycles of 30 s on/30 s off in a Bioruptor Pico (Diagenode) and clarified by centrifugation at 12,000 × g , 4°C for 10 min. SDS-PAGE in Tris-glycine-SDS running buffer and immunoblotting followed standard techniques using the following Abs: mouse monoclonal anti-ISG15 F-9 (catalog no. sc166755; Santa Cruz Biotechnology), rabbit polyclonal anti-MxA (catalog no. 13750-1-AP; Proteintech), goat polyclonal anti-IFIT1 N-16 (catalog no. sc82946; Santa Cruz Biotechnology), mouse monoclonal anti–β-ACTIN, UBA7 (anti-UBE1L B-7; catalog no. sc-390097; Santa Cruz Biotechnology), rabbit monoclonal anti–phosphorylated STAT1 (anti–phospho-STAT1 [Tyr 701 ] 58D6; catalog no. 9167; Cell Signaling Technology), mouse monoclonal anti-PIV5 NP 125 , mouse monoclonal anti-HPIV2 and anti-PIV5 phosphoprotein (P) 161 [Ab cross-reacts with P of both viruses ( )], and mouse monoclonal anti-HPIV3 NP ( ).

Techniques: Virus, Control, Clone Assay, Generated, CRISPR, Infection, Western Blot, Knock-Out, Expressing, Transduction, Gene Knockout