The BMP type I receptor ALK3 is functionally important for hepcidin suppression by ERFE/EPO in primary hepatocytes and in mice. (A-B) Primary hepatocytes were transfected with negative control (Ctrl), Alk2, Alk3, ActrIIa, Actrllb, or Bmpr2 siRNA (80 nM) for 30 hours, followed by treatment with conditioned medium containing 50% (vol/vol) cell supernatant from control HEK293T cells (CTRL-CM) or HEK293T cells overexpressing ERFE (ERFE-CM)²  for 15 hours. Panel A, n = 4 independent experiments; panel B, n = 5 independent experiments. Hepatocyte-specific Alk2-deficient mice (C, Alk2^fl/fl;Alb-Cre), hepatocyte-specific Alk3-deficient mice (D, Alk3^fl/fl;Alb-Cre) and their respective controls (Alk2^fl/fl or Alk3^fl/fl; n = 4-8 per group) at 8 weeks of age were given 1 intraperitoneal dose of EPO (200 U per mouse) or PBS, and livers were collected after 15 hours. Relative hepcidin (Hamp) mRNA levels were determined by using quantitative reverse transcription–polymerase chain reaction. Transcripts were normalized to Rpl19 (in cells) or 18S (in mice) as internal controls, and the average of Ctrl cells treated with CTRL-CM or the respective Alk2^fl/fl or Alk3^fl/fl control mice was set to 1. Data are presented as box plots with minimum to maximum whiskers. *P < .05, **P < .01, ***P < .001 relative to the indicated control by the Student t test or Mann Whitney U test. ns, not significant.