STAT3 is necessary and sufficient to activate the hepcidin promoter downstream of IL-6. HepG2/2.2.1 cells were transiently transfected with a firefly luciferase reporter gene under the control of hepcidin mini, the CE9 mutant or the IL-6 responsive m67 promoter in conjunction with a Renilla luciferase vector with or without constructs expressing mutant STAT3 proteins or siRNA. (A) Constitutively active STAT3 mutant STAT3-C (2.5 μg cDNA). (B) Dominant-negative STAT3 mutant STAT3-DN (2.5 μg cDNA). (C) 2.5 μg STAT3 short hairpin siRNA or vector control. Cells in panels B and C were treated for 12 to 16 hours with 20 ng/mL IL-6. Cell lysates were analyzed for luciferase activity 48 hours after transfection. Relative luciferase activity was calculated as the ratio of firefly to Renilla luciferase activity and is expressed as a multiple of the activity of unstimulated cells transfected with reporter alone. Error bars represent standard deviation of the mean.