Fig 1: Binding sites of the transcription factors NFAT5 and STAT3 on the VEGF-A promoter to induce downstream gene transcription. (A) Transcription Element Search System-based computational analysis of −2,000 bp upstream of the VEGF-A open reading frame. Putative binding domains for NFAT5 and STAT3 at −1,809, −1,471, −840 and −622 bp were predicted to contain consensus sequences on the VEGF-A promoter. MCF-7 cells were transfected with a luciferase reporter driven by constructs of the VEGF-A promoter (−2,000 to +50 bp; mutation *Δ-1,812-11 bp/-2,000 to +50 bp; and mutation *Δ-1,474-73 bp/-2,000 to +50 bp) construct, and stimulated with high salt. (B) MCF-7 cells were transfected with luciferase reporters driven by constructs of the VEGF-A promoter (−2,000 to +50 bp; mutation *Δ-843-42 bp/-2,000 to +50 bp; and mutation *Δ-625-24 bp/-2,000 to +50 bp) construct, and stimulated with IL-17. (C) MCF-7 cells were transfected with luciferase reporters driven by constructs of the VEGF-A promoter (−2,000 to +50 bp; mutation *Δ-1,474-73 bp/-2,000 to +50 bp; mutation *Δ-843-42 bp/-2,000 to +50 bp; or both mutations) construct, and stimulated with high salt and IL-17. Luciferase activity was measured 48 h after transfection and normalized to a Renilla luciferase internal control. The numbers indicate fold-change over the control vector. (D) VEGF-A promoter with binding sites for NFAT5 and STAT3. The four black horizontal bars represent the regions amplified by polymerase chain reaction with specific primers for the −2,000 to −1,751 bp, −1,500 to −1,251 bp, −1,000 to −751 bp and −700 to −450 bp regions of the VEGF-A promoter, and the negative control −250 to +50 bp region of the ACT1a promoter. Chromatin was immunoprecipitated with anti-NFAT5, anti-STAT3 or isotype control immunoglobulin G antibodies from MCF-7 cells following stimulation with both high salt and IL-17. The first three lanes represent chromatin-immunoprecipitation, while the fourth lane represents input chromatin. (E) Co-immunoprecitation of the protein-complex extracted by anti-STAT3 and anti-NFAT5 antibodies, and western blot analysis to probe with the opposite antibody (upper panel, probed with NFAT5 antibody and protein complex pulled-down with anti-STAT3 antibody; lower panel, probed with STAT3 antibody and protein complex pulled-down with anti-NFAT5 antibody). Data are represented as mean values ± standard error of the mean from four independent experiments. Statistical significance was analyzed with the Student's t test, *P<0.05; #P>0.05. Luc, luciferase; IL, interleukin; ACT1a, actin-1a; NFAT, nuclear factor of activated T-cells; STAT, signal transducer and activator of transcription; Ig, immunoglobulin; VEGF, vascular endothelial growth factor.