Activation of the human PF4 promoter by USF1 and USF2. (A) Transient transfection assays were performed with hPF4luc and transcription factor expression vectors. hPF4luc containing 1.7 kb of 5′ flanking region of human PF4 gene in front of the luciferase gene, and 1 μg of pcDNA3 vector, either expressing USF1 or USF2 or both, were cotransfected into HepG2 cells. Relative luciferase activities were measured. The columns and vertical bars represent mean ± SE of 6 replicates. *A significant difference (P < .05) between control and USF1, USF2, and USF1 + USF2. #A significant difference (P < .05) between USF1 and USF2 and USF1 + USF2. (B) Contribution of the E-box motifs in the human PF4 promoter in HEL cells. hPF4mut1, hPF4mut2, and hPF4mut3 contain mutations in the downstream (–234), upstream (–425), or both E-box motifs in hPF4luc. After these plasmids and hPF4luc were transfected into HEL cells, cells were incubated with the medium containing 10 nM PMA, and relative luciferase activities were measured. The columns and vertical bars represent mean ± SE of 12 replicates. *A significant difference (P < .05). (C) The function of E-box motifs in the human PF4 promoter. The same procedure was used as shown in panel B using hPF4(+485)luc and hPF4(+485)Mut. Each construct contains the downstream region from the transcriptional start site in the human PF4 gene. hPF4 (+485)Mut contains mutations in 2 E-box motifs. The columns and vertical bars represent mean ± SE of 6 replicates. *A significant difference (P < .05).