Analysis of the 5'-flanking region of CD164 gene promoter. (A) The PCR products of a series of 5'-deleted mutants of the CD164 gene promoter were subcloned to a pGL3 Luciferase reporter system at the SacI/XholI site, except P1.0 which used a KpnI/XhloI site to generate the CD164 promoter-pGL3 expression plasmid. (B) The 5'-deleted constructs were transfected into HCT116 cells with lipofectamine 2000. 48 hours after transfection, their luciferase activities were examined as described in Methods. Basic pGL3 plasmid without promoter was used as a control. Luciferase activity was normalized to the activity of renilla which were co-transfected as an internal control. The numbers represented the sites 5'-upstream of TSS. Deletion of the region from P2 to P3.2 resulted in a 75% reduction in basal promoter activity. The data bars represented the mean values of three experiments, each performed in triplicate. The error bars represented the standard errors. (C) Relative promoter's activity against promoter's length. P4 showed the optimal activity according to its length. These data suggested the existence of a minimal promoter region within the sequence. (D) Cell-type specificity of CD164 promoter activity. P4 activity was analyzed in different cell lines including K562, HCT116, HT29 and SW480. HCT116 cells showed the highest P4 activity.