Effect of mutations in STAT3-BS or BREs on HAMP promoter response to cytokines. HuH7 cells were transfected with WT-HAMP, mSTAT, or mBRE1/2 promoter-firefly luciferase construct together with pTK-RL construct and treated with the indicated concentration of recombinant human cytokines. (A) IL-6 signaling was abrogated by the mutation in the STAT3-BS, but not by the mutation in both BREs. In contrast, BMP signaling was disrupted by the mutation in both BREs, but not by the STAT3-BS mutation. Bars represent mean ± SD of at least 3 independent experiments executed in duplicate. Statistical significance was determined with the Student t test or Mann-Whitney rank-sum test. *P < .05, compared with untreated control (optimem/WT-HAMP); †P < .05; and ††P < .001, compared with the WT-HAMP construct within the same cytokine group. (B) IL-6 and BMP-9 act synergistically on the HAMP promoter. (C) The mutations in BREs abrogated the synergy, while IL-6 induction was preserved. (D) The mutation of the STAT3-BS did not abrogate the synergy or the BMP-9 induction. Dots and error bars represent mean ± SD. Results are expressed as fold induction over untreated control.