SMAD5 has a dominant role and SMAD1 a contributory role in the regulation of hepcidin in Hep3B cells. Hep3B cells were transfected with negative control (CTRL), SMAD1, SMAD5, or SMAD8 siRNA (20 nM), serum starved with 1% fetal bovine serum overnight, and incubated in the absence or presence of 5 ng/ml of BMP6 for 6 hours. Relative HAMP mRNA levels were determined using quantitative reverse-transcriptase PCR. Transcripts were normalized to an internal control RPL19, and the average of CTRL without BMP6 stimulation was set to 1. Values represent mean ± standard error of the mean. *P < .05; ***P < .001 relative to the respective CTRL by 1-way analysis of variance with Dunnett’s post hoc test (n = 3-4 per group).