Hepcidin is induced by Bmp6 in primary hepatocytes from mice lacking 2 or 3 Smad1/5 alleles but not in double-knockout mice. (A,D-H) Primary hepatocytes were isolated from 6-week-old male Smad5^fl/fl;Cre⁺, Smad1^fl/fl;Cre⁺, Smad1^fl/wt;Smad5^fl/fl;Cre⁺, Smad1^fl/fl;Smad5^fl/wt;Cre⁺, Smad1^fl/fl;Smad5^fl/fl;Cre⁺, and littermate Cre⁻ control mice using 2-step collagenase digestion and cultured in collagen-coated plates. Cells were serum starved with 1% fetal bovine serum overnight before Bmp6 stimulation for 6 hours. Hepatocyte Hamp mRNA levels were determined at baseline (A; n = 4-5 per group) and after stimulation with Bmp6 (D-H; n = 3-5 per group) for each genotype. Transcript levels were normalized to Rpl19, and the average of the respective Cre⁻ control mice without Bmp6 stimulation was set to 1. Values represent mean ± standard error of the mean. *P < .05; **P < .01; ***P < .001 relative to the respective Cre⁻ controls (A) or unstimulated cells of the same genotype (D-H) by Student t test. Fold induction of Hamp mRNA in Bmp6-stimulated versus -nonstimulated cells for each genotype is indicated. (B-C) Smad1 and Smad5 mRNA copy numbers were determined using Taqman primers with probes targeting all known variants and quantitative reverse-transcriptase PCR in mouse primary hepatocytes (B) and human Hep3B cells (C; n = 4 per group). ns, not significant.