Activin B induces hepcidin expression and SMAD1/5/8 phosphorylation in human hepatoma-derived cells. HepG2 cells were treated with IL-6 (50 ng/mL) and/or activin B (50 ng/mL) or activin A (50 ng/mL) for 1.5, 2.5, 4, 6, and 24 hours. (A) Hepcidin (Hamp) mRNA levels were measured by quantitative RT-PCR. Values shown are means of −ΔΔCt (i.e.,−[ΔCt treatment − ΔCt vehicle]) ± SD obtained from 4 independent experiments. Means of−ΔΔCt were compared with 0 by Student t tests. ***P < .001; **P < .01; *P < .05. The fold changes induced by treatment can be obtained by raising 2 to the power −ΔΔCt. (B) Protein extracts were prepared from HepG2 cells treated with the different activators for 2.5 hours. Phospho–Smad1/5/8, total Smad5, phospho–Stat-3, and total Stat-3 were detected by immunoblot techniques. Similar patterns were observed after 4 and 6 hours of treatment (data not shown).