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

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