Figure 3.

Effect of holotransferrin treatment on ERK1/2 activation and of a specific ERK1/2 inhibitor on hepcidin mRNA induction by holotransferrin. (A) After a 42 h period of serum-free culture, hepatocytes were treated either with 30 μM holotransferrin (holoTf) alone (A), 10% serum alone (B), or both (C) for 5, 10, 15, 30, or 120 min. Activation of the ERK1/2 pathway was assessed by immunoblotting 80 μg hepatocyte extracts with the phospho-ERK1/2 antibody. The relative amount of ERK1/2 was ascertained by immunoblot analysis using ERK1/2 antibody. Hepatocytes were treated for 15 min with 50 ng/mL EGF as a positive control for the activation of the ERK1/2 pathway. Molecular weight markers (kDa) are indicated on the left. (D) Relative changes in hepcidin mRNA levels as quantified by real-time qRT-PCR performed on cDNA synthesized from 2 μg total RNA (β-actin normalized, arbitrary units). Hepatocytes were treated for 24 h, after a 42 h period of serum-free culture, with either 0% serum (control cells) or 10% serum with or without 30 μM holoTf, in the presence or absence of 10 μM U0-126 inhibitor added 1 h prior treatment. Results are expressed relative to hepatocytes cultured for 66 h without serum. Mean±SD for four independent samples. The experiment was performed at least three times and a representative result is shown Statistical analysis was performed using Student’s t test (unpaired, two tailed). NS: non significant.