Conserved element 9 mediates IL-6 responsiveness. (A) A diagram of hepcidin mini shows conserved elements (CEs) 1-10 and the luciferase reporter gene (dark gray box). (B) Hep G2/2.2.1 cells were transiently transfected with control plasmid-expressing Renilla luciferase along with one of the following firefly luciferase reporter genes: a 1.3-kb (long) or 0.6-kb (mini) fragment of the 5′ flanking region of the hepcidin gene or a promoterless negative control (basic). At 48 hours after transfection, cells were serum starved for 6 hours and induced with 20 ng/mL IL-6. After 16 hours, firefly luciferase activity (normalized to Renilla luciferase as a transfection control) was measured and compared with activity from uninduced cell lysates. (C) Mutations in hepcidin mini were introduced into CEs 1-4, 6-9, and 10 by site-directed mutagenesis. Each mutant promoter–firefly luciferase plasmid was transiently transfected into HepG2/2.2.1 cells and induced with IL-6 as described in “Materials and methods.” Fold luciferase activity above uninduced controls was compared with that of hepcidin mini. Error bars represent standard error. *P < .001.