Tacrolimus upregulates hepcidin through BMP-SMAD pathway activation. (A) Luciferase activity was analyzed in Hep3B cells transfected with the BRE-Luc reporter vector and treated with tacrolimus (TAC, 1 μg/mL) or vehicle for 15 hours. The luciferase activity of treated cells was normalized to an untreated mean value of 1. A representative experiment, made in triplicate, is shown. (B,C) Hep3B cells, treated with TAC (1 μg/mL), the calcineurin inhibitor cyclosporine A (CA, 1 μg/mL), or vehicle for 15 hours, were processed for RNA purification. Hepcidin (HAMP) and ID1 expression were quantified by qRT-PCR and normalized to the housekeeping gene GAPDH. A representative experiment, made in triplicate, is shown. (D,E) Murine primary hepatocytes were isolated and treated with TAC (1 μg/mL), CA (1 μg/mL), or vehicle for 18 hours. Hepcidin (Hamp) and Id1 expression were evaluated by qRT-PCR and normalized to the housekeeping gene Hprt1. Mean ΔCt values in each group were subjected to a change of origin by subtracting the mean ΔCt of vehicle-treated cells (B: 7.1; C: −3.0; D: 3.0; E: 4.2). A representative experiment, made in triplicate, is shown. Error bars indicate SD. *P < .05; **P < .01; ***P < .001; ****P < .0001. Estimates of the fold changes in gene expression (2^−ΔΔCt) are shown in the graphs.