Cell-free conditioned medium (CM)–dependent hepcidin activation. (A) Conditioned medium (CM) from THP-1 cells was collected after 12, 24, 48, and 72 hours (CM(12h), CM(24h), CM(48h), CM(72h), respectively) in culture. The undiluted CM was used to stimulate Huh7 cells for the indicated time periods. Endogenous hepcidin mRNA expression in Huh7 cells 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 ± SD. (B) Increased luciferase activity after treatment with CM(48h) depends on a functional STAT binding site. Huh7 cells were transfected with the luciferase reporter vectors. Twenty-four hours after transfection cells were treated with CM(48h), and luciferase activity was assessed 24 hours later. Transfections were performed in triplicates, and results are presented as fold change ± SD of firefly/Renilla (F/R). Significant expression changes are marked by an asterisk, whereby * represents P < .05 and ** represents P < .005.