Figure 2

Ferroportin degradation is promoted by BMSC-derived hepcidin in vitro. (A) Human BMSCs were co-cultured with the reporter cell line where ferroportin (the molecule that is responsible for transporting iron from the cells) is tagged with doxycycline-inducible GFP. The ferroportin staining shows a punctate pattern as it is being broken down due to the presence of hepcidin (marked by white arrows). (B) When hepcidin antibody is added to neutralize the effect the dotty pattern disappears and a general membrane staining presents, suggesting the presence of intact FPT in the cells. (C) cultured HEK 293 cells show cytoplasmic staining of FPT-GFP. (D) When SN taken from human BMSCs (previously primed with IL-6) is added to the medium, the cells exhibit a granular, much stronger, cytoplasmic and membrane staining pattern (staining is also visible over the nuclei) that (E) changes back to a cytoplasmic staining (similar to C), after a neutralizing hepcidin antibody is added. (F) The amount of cell nucleus-associated FPT is determined by calculating the ratio of cell nucleus-associated mean green fluorescence intensity values and the cell nucleus area indicated by the blue DNA staining. Eight cells were analyzed in each of the three conditions represented in panels (D, E, F). Friedman test and Dunn’s multiple comparisons test. ***p < 0.001. Ab antibody, FPT ferroportin, GFP green fluorescent protein, ns non-significant, SN supernatant.