IL6-dependent hepcidin activation is mediated by STAT binding motif at position –64/–72 of the hepcidin promoter. (A) Luciferase reporter assays. Huh7 cells were transfected with luciferase reporter vectors and 24 hours later treated with IL6 (1 ng/mL; 24 hours). Transfections were performed in triplicates, and results are presented as a fold change ± SD of firefly/Renilla (F/R). (B) Hepcidin mRNA expression. Huh7 cells were treated with IL6 (1 ng/mL), and endogenous hepcidin mRNA expression was analyzed by quantitative real-time PCR. Data are normalized to mRNA expression of a housekeeping gene, GAPDH, and shown as fold change in comparison with untreated cells. Significant expression changes are marked by an asterisk, whereby * represents P < .05 and ** represents P < .005.