Fig. 1.

A decrease in ferroportin and increase in hepcidin are associated with an increase in the labile iron pool (LIP) in breast cancer cell lines. (A) Concentrations of ferroportin (FPN) in normal and malignant breast cells. Protein (50 μg) from each cell type was analyzed for ferroportin expression by Western blotting. Loading was assessed with an antibody to GAPDH. (B) Hepcidin treatment increases concentrations of ferritin protein in breast cells. HME cells were treated with vehicle and 300 or 700 nM hepcidin for 6 hours, and ferritin H (FH) was assessed by Western blotting. GAPDH was used as a loading control. The increase in ferritin was about twofold as measured by quantification of ferritin/GAPDH ratios by scanning densitometry. (C) Hepcidin treatment increases the LIP. HME cells were treated with 700 nM hepcidin or vehicle control, and the LIP was measured as described in Materials and Methods. rfu, relative fluorescence units. (D) Ferroportin is degraded in normal mammary epithelial (HME) cells treated with hepcidin. Cells were incubated with vehicle and 300 or 700 nM hepcidin for 6 hours, and ferroportin was measured by Western blotting. GAPDH was used as a loading control. (E) Western blot of prohepcidin protein in normal and malignant breast cells. The HepG2 hepatocellular carcinoma cell line was used as a positive control. (Prohepcidin was detected in all cells on prolonged exposure.) β-Actin was used as a loading control. The calculated ratio of prohepcidin to β-actin signal intensity is shown. (F) LIP in normal and malignant breast cells. Graphs show mean and SD of triplicate determinations.