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.