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
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