Tacrolimus increases hepcidin expression in vivo. (A) Schematic representation of the experimental design: C57BL/6 WT male mice (n = 3-6 mice/group) were treated with vehicle or 10 mg/kg TAC via subcutaneous injection and sacrificed at different time points. (B) Liver hepcidin (Hamp) expression and (C) serum hepcidin were quantified by qRT-PCR, as described in supplemental Figure 4A, and competitive enzyme-linked immunosorbent assay, respectively. Hepcidin mRNA was normalized to the housekeeping gene Hprt1 and expressed as a ratio compared with total liver iron content (LIC). (D) Spleen iron content (SIC). Error bars indicate SD. *P < .05; **P < .01. Estimates of the fold changes in gene expression (2^−ΔΔCt) are shown in the graphs.