A human FLK1 promoter-luciferase reporter assay identifies 4 potential NANOG binding sites. (A) Wild-type and point-mutant human FLK1 promoter constructs (a-i). Asterisks indicate point mutations of ATTA to AaTA. (B) Timeline of the transfection experiments and luciferase assay. (C) The indicated wild-type and point-mutant FLK1 promoter constructs driving the luciferase reporter together with a trace amount of β-galactosidase were transiently transfected into HUVECs, which were left untreated (control) or treated with WNT3A (50 ng/mL) (no serum or growth factor). Fold luciferase activity is shown as the mean ± SEM; n = 5-10 from 3-4 independent experiments; *P < .05 and **P < .01 compared with control (without WNT3A treatment). (D-G) Transfection efficiency was determined by staining the transfected HUVECs with X-gal. Magnification, ×100; scale bar, 50 μm. Images were recorded through a Zeiss Axiovert 40 C microscope, 20× objectives, using a Canon Powershot digital camera. Digital images were saved as TIFF documents using Adobe Photoshop CS. Multiple images were assembled using QuarkXpress 8.0 software and labeled, and final images were saved as EPS documents. Experiments were carried out ≥ 3 times.