Journal: Cells

Article Title: The Combination of IFN β and TNF Induces an Antiviral and Immunoregulatory Program via Non-Canonical Pathways Involving STAT2 and IRF9

doi: 10.3390/cells8080919

Figure Lengend Snippet: Experimental design used to study the STAT1-independent delayed transcriptional program induced by the combination of IFNβ and TNF. ( A ) 2ftGH cells were stimulated with either TNF (T), IFNβ (I), or costimulated with IFNβ + TNF (I + T) for 24 h. Quantification of mRNA was performed by qRT-PCR and expressed as fold expression after normalization to the S9 mRNA levels using the ΔΔCt method. Mean +/− SEM, n ≥ 5. Statistical comparison was conducted using one-way ANOVA with Tukey’s post-test. p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), or p < 0.0001 (****). ( B ) U3A (STAT1-deficient), 2ftGH (parental STAT1-positive) cells and U3A-STAT1 cells (U3A cells stably reconstituted with STAT1) were left untreated or stimulated with IFNβ + TNF for the indicated times. WCE (whole cell extracts) were analyzed by SDS-PAGE followed by immunoblot using anti STAT1-P-Tyr701, total STAT1, STAT2-P-Tyr690, total STAT2, IRF9, or actin antibodies. ( C – E ) U3A cells were transfected with siCTRL, siSTAT2, or siIRF9 before being left untreated (NS) or stimulated with IFNβ + TNF for 24 h. ( C ) The schematic describes the workflow of sample preparation and analysis. ( D ) WCE were analyzed by SDS-PAGE followed by immunoblot using anti STAT2, IRF9, and actin antibodies. ( E ) Graph showing the correlation between fold-changes (FC) measured by RNASeq and qRT-PCR for 13 randomly selected genes. Data from siCTRL NS vs. siCTRL IFNβ +TNF, siSTAT2 IFNβ +TNF vs. siCTRL IFNβ + TNF, siIRF9 IFNβ + TNF vs. siCTRL IFNβ + TNF conditions were used.

Article Snippet: U3A cells stably expressing STAT1 were generated by transfection of the STAT1 alpha flag pRc/CMV plasmid (Addgene plasmid #8691; a generous gift from Dr. J. Darnell, Rockfeller University, USA [ , ]) and selection with 800 μg/ml Geneticin (G418).

Techniques: Quantitative RT-PCR, Expressing, Comparison, Stable Transfection, SDS Page, Western Blot, Transfection, Sample Prep

Journal: Cells

Article Title: The Combination of IFN β and TNF Induces an Antiviral and Immunoregulatory Program via Non-Canonical Pathways Involving STAT2 and IRF9

doi: 10.3390/cells8080919

Figure Lengend Snippet: Analysis of STAT1-independent IFNβ + TNF-induced DEGs. ( A ) Diagram describing the bioinformatics analysis strategy used to determine STAT1-independent differentially expressed genes (DEGs) and their regulation by STAT2 and IRF9. ( B ) Volcano plots of the fold-change (FC) vs. adjusted p -value of IFNβ + TNF (Ι + Τ) vs. non-stimulated (NS) siCtrl conditions. ( C ) Volcano plots of the fold-change vs. adjusted p -value of siSTAT2 IFNβ + TNF vs. siCTRL IFNβ + TNF (I + T) conditions. ( D ) Volcano plots of the fold-change vs. adjusted p -value of siIRF9 IFNβ + TNF vs. siCTRL IFNβ + TNF conditions.

Article Snippet: U3A cells stably expressing STAT1 were generated by transfection of the STAT1 alpha flag pRc/CMV plasmid (Addgene plasmid #8691; a generous gift from Dr. J. Darnell, Rockfeller University, USA [ , ]) and selection with 800 μg/ml Geneticin (G418).

Techniques:

Journal: Cells

Article Title: The Combination of IFN β and TNF Induces an Antiviral and Immunoregulatory Program via Non-Canonical Pathways Involving STAT2 and IRF9

doi: 10.3390/cells8080919

Figure Lengend Snippet: Functional characterization of STAT1-independent IFNβ + TNF-induced DEGs. ( A ) Top forty IFNβ + TNF- upregulated DEGs. ( B ) Gene ontology (GO) enrichment analysis of the differentially upregulated genes in IFNβ + TNF vs. non-stimulated siCtrl conditions based on the Biological Processes and Molecular Function categories. Top enriched terms are shown and the full list is available in . ( C ) Modular transcription analysis of upregulated DEGs. Eighteen enriched modules are shown. The full list of enriched modules is available in . ( D ) U3A and STAT1-rescued U3A-STAT1 cells were stimulated with IFNβ (I) or IFNβ + TNF (I + T) for 30 h before infection with VSV at a MOI of 5 for 12 h. The release of infectious viral particles was quantified by plaque forming unit (pfu) assay. The left graphs show dot-plots of all stimulations. Statistical comparisons were performed on the “before and after” plots (displayed on the right of dot-plot graphs) using ratio paired t -tests.

Article Snippet: U3A cells stably expressing STAT1 were generated by transfection of the STAT1 alpha flag pRc/CMV plasmid (Addgene plasmid #8691; a generous gift from Dr. J. Darnell, Rockfeller University, USA [ , ]) and selection with 800 μg/ml Geneticin (G418).

Techniques: Functional Assay, Infection

Journal: Cells

Article Title: The Combination of IFN β and TNF Induces an Antiviral and Immunoregulatory Program via Non-Canonical Pathways Involving STAT2 and IRF9

doi: 10.3390/cells8080919

Figure Lengend Snippet: Analysis of the CXCL10 promoter regulation in response to IFNβ + TNF vs. IFNβ. ( A ) Schematic representation of the CXCL10 promoter (CXCL10prom) luciferase constructs used in this study indicating the main transcription factors consensus binding sites. ( B ) U3A and U3A-STAT1 cells were transfected with the indicated CXCL10prom-luciferase reporter constructs and either left untreated or stimulated with IFNβ or IFNβ + TNF. Relative luciferase activities were measured at 16 h post-stimulation and expressed as fold over the corresponding unstimulated condition. Mean +/− SEM, n = 6. Statistical analyses were performed using an unpaired t-test comparing each promoter to the CXCL10prom-972bp construct. p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****).

Article Snippet: U3A cells stably expressing STAT1 were generated by transfection of the STAT1 alpha flag pRc/CMV plasmid (Addgene plasmid #8691; a generous gift from Dr. J. Darnell, Rockfeller University, USA [ , ]) and selection with 800 μg/ml Geneticin (G418).

Techniques: Luciferase, Construct, Binding Assay, Transfection

Journal: Cells

Article Title: The Combination of IFN β and TNF Induces an Antiviral and Immunoregulatory Program via Non-Canonical Pathways Involving STAT2 and IRF9

doi: 10.3390/cells8080919

Figure Lengend Snippet: Role of distinct STAT2 and/or IRF9-dependent pathways in the regulation of distinct subset of antiviral and immunoregulatory genes in response to IFNβ and TNF. Our data supports a model in which multiple pathways participate to the synergistic action of IFNβ + TNF. While the STAT1-dependent pathway, likely ISFG3, is engaged downstream of IFNβ and TNF, STAT1-independent pathways are also involved in the control of the delayed gene expression. STAT2 and IRF9 act not only in a concerted fashion, likely as a complex, but also independently. IRF9 is known to act as the DNA-binding subunit of the ISGF3 complex and therefore likely mediates binding of STAT2/IRF9 complexes and of alternative complexes devoid of STAT2. The mechanisms of STAT2-dependent regulation of gene expression remains to be characterized.

Article Snippet: U3A cells stably expressing STAT1 were generated by transfection of the STAT1 alpha flag pRc/CMV plasmid (Addgene plasmid #8691; a generous gift from Dr. J. Darnell, Rockfeller University, USA [ , ]) and selection with 800 μg/ml Geneticin (G418).

Techniques: Control, Gene Expression, Binding Assay

Journal: Cell Death & Disease

Article Title: STAT1 β enhances STAT1 function by protecting STAT1 α from degradation in esophageal squamous cell carcinoma

doi: 10.1038/cddis.2017.481

Figure Lengend Snippet: STAT1 β increases and prolongs tyrosine 701 phosphorylation of STAT1 α . ( a ) ESCC cell lines EC1 and KYSE150 were stimulated with IFN- γ , at the doses indicated, or left untreated (w/o) after empty vector or Flag-tagged STAT1 β transfection. Total-protein extracts were used for detection of Tyr701-phosphorylated and total STAT1, flag and STAT1 α by western blotting. ( b ) Both cell lines were stimulated with IFN- γ (10 ng/ml) for the time indicated, or left untreated (w/o) after empty vector or Flag-tagged STAT1β and STAT1α transfection. ( c ) EC1 and KYSE150 cells were stimulated with IFN- γ (10 ng/ml) for the indicated times after empty vector or Flag-tagged STAT1 β and STAT1 α transfection. Phospho-STAT1 was detected with an Alexa Fluor 568-conjugated secondary antibody (red). DAPI (1 μ g/ml) was used for nuclear staining (blue). Fluorescence signals were analyzed with a Zeiss LSM 710 confocal microscope (scale bar 5 μ m). ( d ) Tyrosine phosphorylation of STAT1 α in EC1 cells was detected by immunoprecipitation and western blotting, after empty vector or Flag-tagged STAT1β or STAT1β Y701F plasmid transfection, upon IFN- γ stimulation. Data are representative of three independent experiments

Article Snippet: Plasmids including Flag-STAT1 β and STAT1 α were purchased from Addgene (Cambridge, MA, USA).

Techniques: Phospho-proteomics, Plasmid Preparation, Transfection, Western Blot, Staining, Fluorescence, Microscopy, Immunoprecipitation

Journal: Cell Death & Disease

Article Title: STAT1 β enhances STAT1 function by protecting STAT1 α from degradation in esophageal squamous cell carcinoma

doi: 10.1038/cddis.2017.481

Figure Lengend Snippet: STAT1 β interacts with STAT1 α to protect STAT1 α from proteasome degradation. ( a ) STAT1 α mRNA expression was detected by real-time PCR after transfection of empty vector or Flag-tagged STAT1β or STAT1β Y701F plasmids. Values were normalized to GAPDH and calculated relative to empty vector-transfected cells. Mean values and standard errors (SE) from at least three independent experiments are shown. ( b ) Immunoprecipitation–immunoblotting analysis was performed for STAT1 α and ubiquitination in EC1 cells transfected with empty vector or Flag-tagged STAT1β or STAT1β Y701F plasmids. ( c ) The interaction of STAT1 α and STAT1 β was investigated by immunoprecipitation and western blot analysis in EC1 cells with or without IFN- γ stimulation. Co-immunoprecipitaion was carried out with control IgG and anti-Flag or anti-STAT1 α antibodies as indicated. Immunoprecipitated proteins were analyzed by western blot with anti-STAT1 α and anti-Flag, respectively. ( d ) Co-localization of STAT1 α and STAT1 β in vivo . EC1 and KYSE150 cells were placed on coverslips and stained with the indicated antibodies (scale bar 5 μ m)

Article Snippet: Plasmids including Flag-STAT1 β and STAT1 α were purchased from Addgene (Cambridge, MA, USA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Ubiquitin Proteomics, Control, In Vivo, Staining

Journal: Cell Death & Disease

Article Title: STAT1 β enhances STAT1 function by protecting STAT1 α from degradation in esophageal squamous cell carcinoma

doi: 10.1038/cddis.2017.481

Figure Lengend Snippet: STAT1 β enhances transcription activity and DNA binding of STAT1. ( a ) EC1 and KYSE150 cell lines were transfected with either empty vector, or Flag-tagged STAT1β or STAT1β Y701F plasmids, then stimulated with IFN- γ . After 48 h, the transcription activity of STAT1 was detected by a dual-luciferase reporter assay. ( b ) After transfection with STAT1β or STAT1β Y701F plasmids, the DNA binding ability of STAT1 was detected, in both cell lines, with a STAT1 biotin-probe followed by western blotting. ( c ) Total RNA was extracted and used for RT-qPCR analysis for the genes indicated. GAPDH was used for normalization, and expression levels were calculated relative to empty vector-transfected cells. Triplicate experiments were performed and results from a representative experiment are shown. * P < 0.05; ** P< 0.01

Article Snippet: Plasmids including Flag-STAT1 β and STAT1 α were purchased from Addgene (Cambridge, MA, USA).

Techniques: Activity Assay, Binding Assay, Transfection, Plasmid Preparation, Luciferase, Reporter Assay, Western Blot, Quantitative RT-PCR, Expressing

Journal: Cell Death & Disease

Article Title: STAT1 β enhances STAT1 function by protecting STAT1 α from degradation in esophageal squamous cell carcinoma

doi: 10.1038/cddis.2017.481

Figure Lengend Snippet: Biological functions of STAT1 β in ESCC. ( a ) Tumorigenicity, assessed by colony formation assay, was detected in EC1 and KYSE150 cells, transfected with STAT1β , STAT1β Y701F or empty vector, at different doses of IFN- γ stimulation after 10 days. ( b ) In both cell lines, colony formation was performed in the cells with siRNA knockdown of STAT1 and transfection of STAT1β . ( c ) Using western blot analysis, cleaved and total PARP expression was detected in EC1 and KYSE150 cells transfected with STAT1β or empty vector. ( d ) The chemosensitivity to 5-FU and cisplatin of ESCC cells was assessed by colony formation after transfection of STAT1β for 10 days. ( e ) Cell growth, as assessed by MTS assay, was detected after STAT1β transfection in EC1 and KYSE150 cells after a 4-day exposure to IFN- γ . Triplicate experiments were performed and results from a representative experiment are shown (* P <0.05)

Article Snippet: Plasmids including Flag-STAT1 β and STAT1 α were purchased from Addgene (Cambridge, MA, USA).

Techniques: Colony Assay, Transfection, Plasmid Preparation, Knockdown, Western Blot, Expressing, MTS Assay

Journal: Cell Death & Disease

Article Title: STAT1 β enhances STAT1 function by protecting STAT1 α from degradation in esophageal squamous cell carcinoma

doi: 10.1038/cddis.2017.481

Figure Lengend Snippet: Expression of STAT1 β in ESCC patient samples. ( a ). STAT1 β expression in ESCC tumors was examined by western blot. Compared to benign esophageal tissue harvested at the surgical margins in the same specimens (labeled N), cancerous tissue (labeled Ca) often expressed a lower level of STAT1 β (e.g. cases 1–3). A small subset of tumors (e.g. case 4) had high levels of STAT1 β . ( b ) Immunohistochemistry of formalin-fixed, paraffin-embedded tissues showed variable levels of predominantly cytoplasmic STAT1 β were detectable in esophageal epithelial and ESCC tissues. Based on the staining intensity, normal epithelia and tumors in our cohort were categorized into STAT1-negative, STAT1-weak or STAT1-strong (IHC stain, scale bar, 20 μ m). ( c ) The immunohistochemistry scores of STAT1 β showed that expression of STAT1 β is higher in normal tissues compared to cancer tissues. ( d ) By Kaplan–Meier analysis, a significant correlation between overall survival and the expression level of STAT1 β was found between patients with STAT1 β -strong and STAT1 β -weak/negative staining (* P <0.05)

Article Snippet: Plasmids including Flag-STAT1 β and STAT1 α were purchased from Addgene (Cambridge, MA, USA).

Techniques: Expressing, Western Blot, Labeling, Immunohistochemistry, Formalin-fixed Paraffin-Embedded, Staining, Negative Staining

Journal: Cell Death & Disease

Article Title: STAT1 β enhances STAT1 function by protecting STAT1 α from degradation in esophageal squamous cell carcinoma

doi: 10.1038/cddis.2017.481

Figure Lengend Snippet: Correlation of STAT1 and STAT1 β in 131 ESCC patient samples

Article Snippet: Plasmids including Flag-STAT1 β and STAT1 α were purchased from Addgene (Cambridge, MA, USA).

Techniques: Expressing

Journal: Cell Death & Disease

Article Title: STAT1 β enhances STAT1 function by protecting STAT1 α from degradation in esophageal squamous cell carcinoma

doi: 10.1038/cddis.2017.481

Figure Lengend Snippet: Correlation of STAT1 β expression with ESCC clinical parameters

Article Snippet: Plasmids including Flag-STAT1 β and STAT1 α were purchased from Addgene (Cambridge, MA, USA).

Techniques: Expressing

Journal: Cell Death & Disease

Article Title: STAT1 β enhances STAT1 function by protecting STAT1 α from degradation in esophageal squamous cell carcinoma

doi: 10.1038/cddis.2017.481

Figure Lengend Snippet: Schematic diagram showing STAT1 β interacts with STAT1 α . Upon IFN- γ stimulation, STAT1 can form three different dimers: α : α and β : β homodimers and α : β heterdimers. Dimers can be phosphorylated followed by translocation into the nucleus. Once STAT1 is released from the target gene promoter, it returns to the cytoplasm and is degraded by the proteasome. However, the STAT1 β can bind to STAT1 α to protect STAT1 α from degradation, resulting in prolongation of the half-life of STAT1 α and a concomitant increase in transcription activity

Article Snippet: Plasmids including Flag-STAT1 β and STAT1 α were purchased from Addgene (Cambridge, MA, USA).

Techniques: Translocation Assay, Activity Assay