Effect of heparin on hepcidin stimulation by BMP6 and IL-6. (A) HepG2 cells were incubated for 6 hours with 50 ng/mL IL-6 or 10 ng/mL BMP6, and then hepcidin mRNA level evaluated by quantitative RT-PCR in relationship to HPRT1 mRNA. (B) Same as panel A, except that the cell were first incubated for 16 hours with 200 μg/mL LMWH, and then added of the stimuli and grown in the presence of heparin for 6 hours. (C) Same as panel B, except that the cells were washed before addiction of the stimulating factors, and grown for 6 hours in the absence of heparin. (D) The cells were grown for 6 hours in the presence or absence of 10 ng/mL BMP6, then added UFH 4 μg /mL and grown for another 16 hours. The schemes of the experiments are represented above the graphs, where stimuli is the incubation with IL-6 or BMP6, and heparin treatment is with 200 μg/mL LMWH (A-C) or with 4 μg/mL UFH (D). Bottom: Western blotting of phosphorylated STAT3 (pSTAT3), of phosphorylated SMAD1/5/8 (pSMAD1/5/8), total SMAD5 and of β-actin as calibrator representative of each panel A, B, C, and D, respectively. The real-time RT-PCR data are means and SD of 2 independent experiments in triplicate. The asterisks indicate significant difference (P < .05) from the nonstimulated control. Western blots are representative of at least 2 independent experiments. The vertical solid lines separate lanes of the same electrophoresis experiment that were spliced out in the figure preparation to eliminate irrelevant or uninformative lanes.