Fig. 2. H-ferritin immunostaining in SW1088 human astrocytoma cells is not regulated by DNA synthesis but does change in response to iron chelation. H-ferritin was detected with the HO2 monoclonal mouse anti-human rH-ferritin antibody (A,C,E,G,I). The sections were colocalized with DAPI in order to visualize the nucleus (B,F,H,J) or BrdU for DNA synthesis (D). (A-D) Cells that have been triple-stained for H-ferritin (A,C), DAPI (B) and BrdU incorporation (D). (A,B) H-ferritin is detected in the nucleus of astrocytoma cells grown in standard media. The arrow indicates an example of a single cell nucleus. The cell nuclei from A are shown in B using DAPI. The arrow indicates the same cell in A and B. (C,D) A is reproduced as C in order to compare H-ferritin staining with that of BrdU (D). H-ferritin is found in the nuclei of both BrdU and non-BrdU positive cells. (D) The arrows in C,D indicate the nucleus of the same cell that is both H-ferritin and BrdU positive. The asterisk near the cell in C indicates a cell that has H-ferritin in the nucleus but is not BrdU positive. (E,F) H-ferritin is detected in the nucleus and cytoplasm of astrocytoma cells (E) but is not localized to the nucleus in dividing cells (F). The DAPI stain in F reveals two cells (arrows) that are dividing and the area containing the chromosomes is not visible in those cells in E. (G,H) H-ferritin is only cytoplasmic in most (>85%) DFO-treated cells. The nuclei of the cells in G are demonstrated using DAPI (H) and the arrows depict a cell in which nuclear ferritin is not detected. (I,J) H-ferritin is detected in the nucleus after being returned to standard media after DFO treatment (I). The arrows indicate the nucleus of a cell that is stained for H-ferritin (I) and DAPI (J). Bar, 10 µm.

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