Journal: Scientific Reports

Article Title: Amelioration of Experimental autoimmune encephalomyelitis and DSS induced colitis by NTG-A-009 through the inhibition of Th1 and Th17 cells differentiation

doi: 10.1038/s41598-018-26088-y

Figure Lengend Snippet: NTG-A-009 reduces Th1 and Th17 cells differentiation through the inhibition of JAK/STAT pathway. ( a ) Naïve CD4 + T cells isolated from spleen and lympnodes were cultured with or without NTG-A-009 under Th1 condition and re stimulated with anti-CD3 (1 μg/ml) and anti-CD28(1 μg/ml) followed by the analysis of the expression of indicated proteins in JAK/STAT pathway by western blot. Phosphorylated and total form of STAT1, STAT4, and JAK1/2 were analyzed by immunoblotting under Th1 condition. ( b ) Phosphorylated and total form of STAT3 and JAK1/2 were detected by immunoblotting under Th17 condition. Full length blots and gels were shown in supplementary information- 7, 8, 9 and 10. Data are the representative of three independent experiments.

Article Snippet: Cells were then transfected with an STAT3 (pcDNA3-STAT3-Y705F, addgene plasmid) and STAT1 expressing plasmid (STAT1 alpha Y701F pRc/CMV, addgene plasmid) using fugene HD transfection reagent (Promega Madison, USA) as described by manufacturer protocol.

Techniques: Inhibition, Isolation, Cell Culture, Expressing, Western Blot

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