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: Molecular cancer

Article Title: The homeoprotein DLX4 controls inducible nitric oxide synthase-mediated angiogenesis in ovarian cancer.

doi: 10.1186/s12943-015-0368-3

Figure Lengend Snippet: Figure 3 DLX4 stimulates STAT1 activity and induces iNOS expression in a STAT1-dependent manner. (A) qRT-PCR analysis of relative NOS2 mRNA levels in vector-control ES2 cells and in ES2 cells that expressed wild-type DLX4 or mutant DLX4 (DLX4-TA) with or without dominant-negative STAT1 (STAT1-dn). (B) Vector-control ES2 cells and ES2 cells that expressed wild-type or mutant DLX4 were transfected with a firefly luciferase reporter construct driven by GAS elements (GAS-LUC), stimulated without or with IFN-γ (10 ng/mL) for 16 h and then assayed for luciferase activity. (C) Activity of the GAS-LUC reporter construct was assayed in 2008 cells that expressed non-targeting and DLX4 shRNAs as described in (B). (D) Western blot analysis of levels of total STAT1 and phosphorylated STAT1 in vector-control and +DLX4 ES2 cells that were stimulated with IFN-γ (10 ng/mL) for 0, 1, 6 and 18 h. (E) Lysates of U3A cell lines that lacked or stably expressed GFP-STAT1 fusion protein and/or FLAG-tagged DLX4 fused to GFP were assayed by Western blot using Abs to STAT1 and DLX4. (F) Activity of the GAS-LUC reporter construct in STAT1-deficient U3A cells and in U3A cells reconstituted with STAT1 that lacked or expressed DLX4. Transfected cells were stimulated with or without IFN-γ (10 ng/mL) for 16 h and then assayed for luciferase activity. (G) FLAG Ab was used to pull down FLAG-tagged DLX4 in U3A cells that were stimulated with IFN-γ (10 ng/mL) for 1 h. Immunoprecipitates were analyzed by Western blot using Ab to STAT1. Pulldown using control Ig was included as a negative control. Shown in B, C and F are relative firefly luciferase activities in three independent experiments.

Article Snippet: Other plasmids were as follows: pGFP-V-RS plasmids containing non-targeting and DLX4 shRNAs (OriGene Technologies), pGIPZ plasmids containing NOS2 shRNAs (GE Healthcare), eGFP-STAT1 (provided by Alan Perantoni, National Cancer Institute, Frederick, MD; Addgene plasmid 12301) [48], pRc/CMV-Flag STAT1α Y701F [28] (provided by James Darnell, Rockefeller University, New York, NY; Addgene plasmid 8702).

Techniques: Activity Assay, Expressing, Quantitative RT-PCR, Plasmid Preparation, Control, Mutagenesis, Dominant Negative Mutation, Transfection, Luciferase, Construct, Western Blot, Stable Transfection, Negative Control