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: Nature Communications

Article Title: PARP9 and PARP14 cross-regulate macrophage activation via STAT1 ADP-ribosylation

doi: 10.1038/ncomms12849

Figure Lengend Snippet: ( a ) The consequences of PARP9 and PARP14 silencing on IFNγ stimulated (TNFα, IL-1β and CCL2/MCP-1) and IL-4 stimulated (MRC1) gene expression in human primary macrophages ( n =8). ( b ) The consequences of PARP9 and PARP14 silencing on IFNγ stimulation (TNFα and iNOS) and IL-4 stimulation (Arg1 and MRC1) gene expression in mouse bone marrow-derived macrophages ( n =3). ( c ) The ratio of phosphorylated STAT1 and STAT6 protein levels to total STAT1 and STAT6 (pSTAT1/tSTAT1 ratio and pSTAT6/tSTAT6 ratio) in human primary macrophages ( n =6 and n =5, respectively) of the PARP9 and PARP14 silencing experiments. * P <0.05 and ** P <0.01, respectively, by Student's t -test. Error bars indicate s.d.

Article Snippet: Primary antibodies against human and mouse PARP14 (1:250, HPA01206 Sigma-Aldrich), human and mouse PARP9 (1:250, ab53796, Abcam), human and mouse STAT1 (1:1,000, #9172, Cell Signaling), phosphorylated STAT1 (1:1,000, #9167, Cell Signaling), human and mouse STAT6 (1:2,000, #9362, Cell Signaling), mouse (1:1,000, ab54461, Abcam) and human (1:2,000, #9361, Cell Signaling) phosphorylated STAT6 and human and mouse β-actin (1:5,000; Novus) were used.

Techniques: Gene Expression, Derivative Assay

Journal: Nature Communications

Article Title: PARP9 and PARP14 cross-regulate macrophage activation via STAT1 ADP-ribosylation

doi: 10.1038/ncomms12849

Figure Lengend Snippet: ( a ) PARP14 silencing and enforced expression significantly affected PARP9 gene expression in IFNγ-stimulated THP-1 cells ( n =3). PARP9 silencing increased PARP14 gene expression ( n =3). ( b ) Co-IP assay revealed a complex between PARP9 and PARP14. ( c ) Intracellular colocalization of PARP9 and PARP14 in the cytosol in M(-) and M(IFNγ). ( d ) PARP9 inhibits ADP-ribosylation of STAT1α and STAT6 by PARP14 (protein ribosylation assay). PARP14 auto-ribosylation is also indicated. * P <0.05 and ** P <0.01, respectively, by Student's t -test. Error bars indicate s.d.

Article Snippet: Primary antibodies against human and mouse PARP14 (1:250, HPA01206 Sigma-Aldrich), human and mouse PARP9 (1:250, ab53796, Abcam), human and mouse STAT1 (1:1,000, #9172, Cell Signaling), phosphorylated STAT1 (1:1,000, #9167, Cell Signaling), human and mouse STAT6 (1:2,000, #9362, Cell Signaling), mouse (1:1,000, ab54461, Abcam) and human (1:2,000, #9361, Cell Signaling) phosphorylated STAT6 and human and mouse β-actin (1:5,000; Novus) were used.

Techniques: Expressing, Gene Expression, Co-Immunoprecipitation Assay

Journal: Nature Communications

Article Title: PARP9 and PARP14 cross-regulate macrophage activation via STAT1 ADP-ribosylation

doi: 10.1038/ncomms12849

Figure Lengend Snippet: ( a ) The amino-acid sequence of human STAT1α C terminus. Green amino acids indicate ribosylated peptides; confirmed ribosylation sites are underlined. STAT1 is phosphorylated at indicated tyrosine (red). ( b ; Left panels) MS/MS spectra for the mono-ADP-ribosylated peptides and corresponding unmodified forms. ADP-ribose fragments are annotated in green. *, ribosylation site; m, oxidized methionine. The grey circles indicate background or undetermined ions. (Right panels) MS1-based quantification of PARP9 inhibition of PARP14-mediated STAT1α ribosylation at E657 (upper panel) and E705 (lower panel), respectively. ( c ) Effects of mutated amino acids at E657 and E705 in STAT1 (ribosylation sites for PARP14) on its Tyr701 phosphorylation and pro-inflammatory gene expression in mouse bone marrow-derived macrophages ( n =4). * P <0.05 and ** P <0.01, respectively, by Student's t -test. Error bars indicate s.d.

Article Snippet: Primary antibodies against human and mouse PARP14 (1:250, HPA01206 Sigma-Aldrich), human and mouse PARP9 (1:250, ab53796, Abcam), human and mouse STAT1 (1:1,000, #9172, Cell Signaling), phosphorylated STAT1 (1:1,000, #9167, Cell Signaling), human and mouse STAT6 (1:2,000, #9362, Cell Signaling), mouse (1:1,000, ab54461, Abcam) and human (1:2,000, #9361, Cell Signaling) phosphorylated STAT6 and human and mouse β-actin (1:5,000; Novus) were used.

Techniques: Sequencing, Tandem Mass Spectroscopy, Inhibition, Phospho-proteomics, Gene Expression, Derivative Assay

Journal: Nature Communications

Article Title: PARP9 and PARP14 cross-regulate macrophage activation via STAT1 ADP-ribosylation

doi: 10.1038/ncomms12849

Figure Lengend Snippet: ( a–c ) Cultured peritoneal macrophages derived from PARP14 −/− and PARP14 +/+ mice. ( a ) IFNγ and IL-4 pathway gene expression profiles ( n =3). ( b ) Secretion of inflammatory factors into culture media ( n =3). ( c ) Western blot and corresponding densitometry quantification of phosphorylated STAT1 and STAT6. Each data point is the average of triplicate samples per donor ( n =3). ( d ) Left: representative images of haematoxylin and eosin (H&E; top) and Mac3 (bottom) staining. Scale bars, 100 μm. Right: quantification of lesion formation in mechanically injured femoral arteries of PARP14 −/− and PARP +/+ mice. Mac3 staining represents macrophage accumulation ( n =4–5). ( e ) LCM of the neointima followed by gene expression analysis ( n =4). ( f ) Flow cytometry analysis of splenic CD11b+Ly6G− monocytes after induction of mechanically injured femoral arteries of PARP14 +/+ and PARP14 −/− mice ( n =3). ( g ) Representative H&E staining images and quantification of neointima formation in mechanically injured femoral arteries after bone marrow transplantation (BMT) PARP14 +/+→+/+ and PARP14 −/−→+/+ mice ( n =6). Scale bars, 100 μm. * P <0.05 and ** P <0.01, respectively, by Student's t -test. Error bars indicate s.d.

Article Snippet: Primary antibodies against human and mouse PARP14 (1:250, HPA01206 Sigma-Aldrich), human and mouse PARP9 (1:250, ab53796, Abcam), human and mouse STAT1 (1:1,000, #9172, Cell Signaling), phosphorylated STAT1 (1:1,000, #9167, Cell Signaling), human and mouse STAT6 (1:2,000, #9362, Cell Signaling), mouse (1:1,000, ab54461, Abcam) and human (1:2,000, #9361, Cell Signaling) phosphorylated STAT6 and human and mouse β-actin (1:5,000; Novus) were used.

Techniques: Cell Culture, Derivative Assay, Gene Expression, Western Blot, Staining, Flow Cytometry, Transplantation Assay

Journal: Journal of Immunotherapy

Article Title: Adoptive Transfer of Interleukin-21-stimulated Human CD8+ T Memory Stem Cells Efficiently Inhibits Tumor Growth

doi: 10.1097/cji.0000000000000229

Figure Lengend Snippet: FIGURE 4. IL-21 only highly upregulates the phosphorylation of STAT3 in the process of TSCM priming. Naive CD8+ T cells display identical STAT phosphorylation profiles in response to IL-2 or IL-21. FACS-purified CD8+ T cells from healthy donors were activated with coated anti-CD3 (2 μg/mL) and soluble anti-CD28(1 μg/mL) overnight, then stimulated with 10 ng/mL IL-2 or 20 ng/mL IL-21. Two hours after stimulation, cells were lysed in sodium dodecyl sulfate polyacrylamide gel electrophoresis sample buffer. Equivalent amounts of proteins were analyzed by Western blot for indicated phosphoproteins. Data in STAT1 (A) STAT3 (B) STAT5 (C) Akt (D) and Erk1/2 (E) are shown representative of 3 experiments. IL-21 indicates interleukin-21; STAT, signal transducer and activator of transcription; TSCM, T memory stem cell.

Article Snippet: Total protein (20 μg) was separated on a 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis gel followed by standard immunoblotting with primary antibodies for Stat1, phospho-Stat1 (Tyr701), Stat3, phospho-Stat3 (Tyr705, Ser727), Stat5, phospho-Stat5 (Tyr694), Akt, phospho-Akt (Ser473), Erk1/2, phospho-Erk1/2 (Thr202/Tyr204) (Cell Signaling Technology, Danvers, MA), and GAPDH (Proteintech, Wuhan, China) or α-tubulin (Cell Signaling Technology), followed by fluorescence-conjugated goat anti-mouse or antirabbit IgG (LI-COR, Lincoln).

Techniques: Phospho-proteomics, Purification, Polyacrylamide Gel Electrophoresis, Western Blot

Journal: Journal of Neuroinflammation

Article Title: The histone deacetylase inhibitor suberoylanilide hydroxamic acid attenuates human astrocyte neurotoxicity induced by interferon-γ

doi: 10.1186/1742-2094-9-113

Figure Lengend Snippet: Effects of SAHA on IFN-γ-induced phosphorylation of STAT1 and STAT3 in human astrocytes. Human astrocytes were incubated with or without SAHA at the concentrations indicated for 1 h. The cells were subsequently stimulated with IFN-γ for 30 minutes. Astrocytes in the control group were incubated with medium only. Cell lysates were separated by 8% SDS-PAGE and immunoblotted for phospho-Tyr 701 -STAT1 (pSTAT1) and total STAT1 (A) or phospho-Tyr 705 -STAT3 (pSTAT3) and total STAT3 (C) . The density ratios of phosphorylated to total protein are shown as mean ± S.E.M. of three independent experiments (B, D) . *Significantly different from IFN-γ stimulation only.

Article Snippet: The protein was transferred to a PVDF membrane at 70 V for 2 h. The membrane was blocked with 5% skim milk plus 3% bovine serum albumin (BSA) in PBS at room temperature (RT) for 1 h. Subsequently, the membrane was incubated with specific rabbit antibodies against phospho-Tyr 701 -STAT1 (1:2,000), total STAT1 (1:1,000), phospho-Tyr 705 -STAT3 (1:2,000) or total STAT3 (1:1,000) at 4°C overnight and then treated with horseradish peroxidase-conjugated anti-rabbit IgG antibody (1:2,000) at RT for 1 h. All antibodies used for immunoblotting were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Phospho-proteomics, Incubation, Control, SDS Page

Journal: PLoS ONE

Article Title: An Autocrine Cytokine/JAK/STAT-Signaling Induces Kynurenine Synthesis in Multidrug Resistant Human Cancer Cells

doi: 10.1371/journal.pone.0126159

Figure Lengend Snippet: A. The cDNA from A549 and A549/dx cells was analyzed by a PCR array specific for JAK/STAT signaling, as reported under Materials and methods. The fold regulation of the 83 genes analyzed, expressed in logarithmic scale, is represented in a colorimetric scale. The figure is the mean of 4 experiments. B. The cells were lysed and subjected to the Western blot analysis for phospho(Tyr 1022/1023)-JAK1, JAK1, phospho(Tyr701)-STAT1, STAT1, phospho(Tyr705)-STAT3, STAT3. The β-tubulin expression was used as control of equal protein loading. The figure is representative of 3 experiments with similar results. C. The expression of PIAS1, PIAS3, phospho(Tyr701)-STAT1, STAT1, phospho(Tyr705)-STAT3, STAT3 in nuclear extracts was measured by Western blotting. The TBP expression was used as control of equal protein loading. The figure is representative of 3 experiments with similar results.

Article Snippet: Nuclear extracts were prepared with the Nuclear Extract Kit (Active Motif, Rixensart, Belgium); 10 μg of nuclear proteins were resolved by SDS-PAGE and probed with the following antibodies against: PIAS1 (rabbit monoclonal, diluted 1:1,000, ab109388, Abcam); PIAS3 (rabbit polyclonal, diluted 1:1,000, ab22856, Abcam); phospho(Tyr701)-STAT1; STAT1; phospho(Tyr705)-STAT3; STAT3; TATA-binding protein (TBP; rabbit polyclonal, diluted 1.500, sc-273, Santa Cruz Biotechnology Inc.).

Techniques: Western Blot, Expressing, Control

Journal: PLoS ONE

Article Title: An Autocrine Cytokine/JAK/STAT-Signaling Induces Kynurenine Synthesis in Multidrug Resistant Human Cancer Cells

doi: 10.1371/journal.pone.0126159

Figure Lengend Snippet: A549/dx cells were grown for 48 h in fresh medium (CTRL), treated with a non-targeting scrambled siRNA (scr) or with a specific siRNAs pool targeting STAT1 or STAT3, respectively (si STAT1, si STAT3). Untreated chemosensitive A549 cells were used as control. A . The expression of STAT1, STAT3, IDO1, IDO2 and TDO was measured in whole cell lysates by Western blotting, 48 h after the transfection. The β-tubulin expression was used as control of equal protein loading. The figure is representative of 3 experiments with similar results. B . The kynurenine levels in the cell culture supernatants were measured spectrophotometrically. Data are presented as means ± SD (n = 4). * p < 0.01: versus A549 CTRL; ° p < 0.005, °° p < 0.001: versus A549/dx CTRL. C . The proliferation of activated T-lymphocytes collected from PBMC after a 72 h co-incubation with A549 and A549/dx cells was measured with the [ 3 H]thymidine assay. In the presence of anti-CD3 and anti-CD28 stimulatory antibodies without tumor cells (positive control), the [ 3 H]thymidine incorporation was 28,926 ± 1,426 cpm; in the presence of RPMI medium alone (negative control), the [ 3 H]thymidine incorporation was 4,312 ± 529 cpm. Data are presented as means ± SD (n = 6). * p < 0.05: versus A549 CTRL; ° p < 0.01, °° p < 0.005: versus A549/dx CTRL. D . The percentage of CD3 + T-lymphocytes collected from PBMC, co-incubated with tumor cells as reported in C , was measured by flow cytometry. Data are presented as means ± SD (n = 6). * p < 0.01: versus A549 CTRL; ° p < 0.01, °° p < 0.002: versus A549/dx CTRL.

Article Snippet: Nuclear extracts were prepared with the Nuclear Extract Kit (Active Motif, Rixensart, Belgium); 10 μg of nuclear proteins were resolved by SDS-PAGE and probed with the following antibodies against: PIAS1 (rabbit monoclonal, diluted 1:1,000, ab109388, Abcam); PIAS3 (rabbit polyclonal, diluted 1:1,000, ab22856, Abcam); phospho(Tyr701)-STAT1; STAT1; phospho(Tyr705)-STAT3; STAT3; TATA-binding protein (TBP; rabbit polyclonal, diluted 1.500, sc-273, Santa Cruz Biotechnology Inc.).

Techniques: Control, Expressing, Western Blot, Transfection, Cell Culture, Incubation, Positive Control, Negative Control, Flow Cytometry