Fig. 1.

Response of hepatocyte hepcidin levels to serum: (a–b) Primary hepatocytes were treated in serum-free media (Optimem) with 0 or 5% human serum or plasma overnight, then harvested and analyzed by Northern blot for hepcidin, albumin and β-actin RNA levels. (c) 1 mL human serum, diluted four-fold with 20 mM Tris pH 8.0, was fractionated with a 5 mL column volume (CV) GE HiTrap Blue column with high affinity for albumin; flowthrough (FT) and eluate (EL) fractions were collected and concentrated and desalted to equal volumes with 5 kD molecular weight cut-off centrifugal filtration devices. Hepatocytes were treated overnight with equal volumes of FT and EL fractions; RNA was isolated and analyzed for β-actin and hepcidin mRNA levels. (d) 1 mL human serum was diluted and chromatographed with GE HiTrap Blue (5 mL CV) and Q anion exchange (1 mL CV) columns. Fractions were eluted from the Q column with 75, 150 and 300 mM NaCl and concentrated and desalted using centrifugal filtration devices. Hepatocytes were treated overnight with 0 or 5% serum or equal volumes of anion exchange fractions. RNA was isolated and analyzed by Northern blot. Band intensity was quantitated by NIH Image software and expressed as a ratio of hepcidin to β-actin mRNA band intensity per μg of fraction protein. (e) Fractions from (d) were subjected to denaturing, reducing SDS PAGE and Coomassie staining. (f) Hepatocytes were treated in serum-free media with 0 or 2% human serum and 0 or 10% concanavalin A equilibration (20 mM Tris pH 7.4/0.5 M NaCl/5 mM MgCl₂, MnCl₂, CaCl₂) or elution buffer (500 mM methyl-α-D-glucopyranoside) overnight, then analyzed for hepcidin and β-actin expression levels by Northern blot. (g) Hepatocytes were incubated in serum-free media with or without 50 μM MnCl₂, CaCl₂ or MgCl₂ overnight, then analyzed as in (f). Results in all panels are representative of three independent experiments