Impact of BMP signaling on IL-6–mediated regulation of hepcidin. HepG2 cells were pretreated with varying concentrations of LDN-193189 for 30 minutes and (A) stimulated with IL-6 (100 ng/mL) or (B) vehicle for 90 minutes. Hepcidin mRNA levels were measured by qRT-PCR (values are mean ± SEM, n ≥ 3, 1-way ANOVA P < .05, *P < .05 vs untreated control, #P < .05 vs IL-6–treated control). (C) HepG2 cells were pretreated with and without LDN-193189 (100nM) for 30 minutes and stimulated with or without IL-6 (100 ng/mL). Hepcidin mRNA levels were measured by qRT-PCR (1-way ANOVA P = .01, *P = .01 vs control, #P < .05 vs control, and †P < .02 vs IL-6–stimulated cells). (D) HepG2 cells were pretreated with noggin (1 μg/mL), ALK3-Fc (1 μg/mL), or vehicle for 30 minutes and incubated with IL-6 (100 ng/mL) or vehicle for 90 minutes, and hepcidin mRNA levels were measured by qRT-PCR (n = 3, 1-way ANOVA P < .0001, *P < .04 vs vehicle-treated controls, #P < .05 vs IL-6–treated controls). (E) HepG2 cells were treated with either vehicle, BMP6 (2.5 ng/mL), IL-6 (100 ng/mL), or BMP6 and IL-6, and hepcidin mRNA levels were measured by qRT-PCR (n = 3, 1-way ANOVA P = .003, *P < .05 vs untreated control, #P < .05 vs IL-6–treated control). (F) Protein extracts were prepared from HepG2 cells treated either with vehicle, BMP6 (10 ng/mL), IL-6 (100 ng/mL), or BMP6 and IL-6 for 30 minutes. Phosphorylated SMAD1/5/8 and total SMAD1 were detected by immunoblot techniques.