The functions of overlapping E-box, HIF-1alpha and AP-1-like sequences within CD164 P4 promoter. (A) Schematic presentation of the Overlapping HIF-1A/E-box/AP-1-like in the minimal promoter region of CD164. (B) Mutation analysis of the putative E-box on CD164 promoter P4 region. Constructs with mutation of the E-box in the promoter region were subcloned into the pGL3-basic plasmid and the HCT116 cells were transfected. Significant decrease of luciferase activity was found in the E-box mutation P4 mutant (p < 0.001, Student's t-test). Co-transfection of a HIF-1alpha expressing plasmid with pGL3P4 resulted in a significant increase in the promoter activity (p < 0.01, Student's t-test). To investigate the effect of HIF-1alpha through HIF-1alpha/E-box overlapping sequence, the pcDNA3.1- HIF-1alpha was co-transfection with PGL3P4 containing E-box mutation. For the normalized luciferase activity, a significant decrease of luciferase activity was found in the co-transfection with PGL3P4 containing E-box mutation compared to the HCT116 cells co-transfected with wild-type PGL3P4 (p < 0.001, student T-Test).