Smad1 and Smad5 are required for EPO and erythroferrone suppression of hepcidin in mice.Smad1^fl/fl;Smad5^fl/fl;Cre⁺ and littermate Cre⁻ mice at 6 weeks of age were injected with phosphate-buffered saline (PBS) or EPO (200 U per mouse), and tissues were harvested after 15 hours to determine bone marrow Fam132b mRNA expression (A), liver Hamp mRNA (B), liver phosphorylated Smad1/5 protein (C), and liver Id1 mRNA levels (D). Primary hepatocytes isolated from 6-week-old male Smad1^fl/fl;Smad5^fl/fl;Cre⁺ and littermate Cre⁻ mice (E-F) and Hep3B cells (G-I) were treated with conditioned medium containing 50% (volume-to-volume ratio) cell supernatant from control HEK293T cells (Ctrl-CM) or Erfe-CM for 15 hours (E-F) or 6 hours (G-I), and the relative mRNA levels of Hamp (E,G), Id1 (F,H), and pSMAD1/5 protein (I) were determined. (J) Hep3B cells were transfected with control siRNA or SMAD1 and SMAD5 siRNA for 48 hours before 6-hour Ctrl-CM or Erfe-CM treatment, and relative HAMP mRNA levels were determined. Transcript levels measured by quantitative reverse-transcriptase PCR were normalized to Rpl19, Smad1/5 phosphorylation levels determined by immunoblot were normalized to total Smad5, and the average of PBS-treated Cre⁻ control mice or Ctrl-CM–treated cells was set to 1. Representative immunoblots are shown. Values represent mean ± standard error of the mean (n = 4-5 mice per group in panel A; n = 10 mice per group in panels B-D; n = 4 per group in panels E-F; n = 3 per group in panels G-J). *P < .05; **P < .01; ***P < .001 relative to PBS-treated mice or Ctrl-CM–treated cells of the same genotype by Student t test. ns, not significant.