Effect of heparin on hepcidin expression and BMP signaling in HepG2 cells. (A) The cells were grown for 16 hours in the absence of exogenous BMPs with the indicated concentrations of heparin: UFH, LMWH, and fondaparinux pentasaccharide, and the hepcidin mRNA levels were quantified by real-time RT-PCR in relationship to HPRT1 mRNA. The data are expressed in a logarithmic scale; the inset shows the inhibition plot of UFH in a linear scale. Data are means and SD of 3 independent experiments in triplicate. (B) Western blotting of phosphorylated SMAD1/5/8, total SMAD5, and of β-actin as calibrator of the cell extracts after the incubation with UFH and with fondaparinux. Data are from 2 independent experiments in triplicate. (C) Cells were incubated for the indicated time with 200 μg/mL LMWH, and the level of hepcidin mRNA were quantified by real-time RT-PCR. Data are representative of 4 independent experiments.