MMB inhibits SMAD1/5/8 phosphorylation and hepcidin expression in vitro and in vivo. (A) Quantitative reverse transcription polymerase chain reaction analysis of Hamp mRNA levels in HepG2 cells stimulated with 10-ng/mL BMP6 for 6 hours. Results are expressed as percentages of hepcidin induction normalized to vehicle-treated cells (50% effective concentration: MMB = 0.65 µM; RUX > 10µM; n = 3). (B) Representative western blots of pSMAD1/5/8 and pSTAT3 levels after 30-minute stimulation with 10-ng/mL BMP6 (for pSMAD1/5/8 induction) or 20-minute stimulation with 10-ng/mL IL-6 (for pSTAT3 induction; top and middle). β-actin was used as a loading control. Compound (cpd) was always added 2 hours before BMP6 stimulation. Percentages of pSMAD1/5/8/pSTAT3 induction (normalized to vehicle control-treated cells; n = 5 for pSMAD1/5/8; n = 2 for pSTAT3) ± standard errors of the mean (bottom). (C) Representative western blot and quantification (n = 5 per group) of hepatic nuclear extracts for pSTAT3 and pSMAD1/5/8 from ACD rats treated for 3 days with MMB (5, 10, or 25 mg/kg). TATA-binding protein (TBP) and STAT3 levels were used for normalization. Each lane represents one individual animal. Analysis of variance with Dunnett’s correction for multiple comparisons vs ACD was applied (C). Results are shown as means ± standard errors of the mean. *P < .05, ***P < .001, ****P < .001.