C/EBPα mediates a high level of basal hepcidin mRNA expression. (A) Luciferase reporter assays. Huh7 cells were transfected with luciferase reporter vectors, and luciferase activity was measured after 72 hours. Transfections were performed in triplicates, and results are presented as fold change ± SD of firefly/Renilla (F/R). The –165 bp promoter construct (lacking the C/EBPα binding motif) shows more than 3-fold reduced luciferase activity compared with the –942 bp and –385 constructs. Luciferase activity of the –942 bp construct was set to 100%. (B) siRNA-mediated knockdown of C/EBPα. Hepcidin mRNA expression in Huh7 cells was assayed after transfection with specific siRNA directed against C/EBPα or luciferase (Luc siRNA) as a control. Hepcidin and C/EBPα mRNA expression was analyzed by quantitative real-time PCR, and data were normalized to mRNA expression of a housekeeping gene, GAPDH. Data are presented as fold change whereby Huh7 cells transfected with the luciferase control siRNA were set to 100%. Significant expression changes are marked by an asterisk, whereby * represents P < .05 and ** represents P < .005.