Fig. 4. Ferritin nuclear translocation in SW1088 human astrocytoma cells responds to iron chelation treatment and is ferritin-subunit specific. These are a series of confocal images that are shown for each condition as a representative of the entire cell population. Each experiment was repeated three times and random cells chosen. The arrow in each panel points to the cell nucleus. Fluorophore-labeled proteins (5 µM) were added to permeabilized cells for 60 minutes prior to visualization. (A) TRITC-dextran (70,000 kDa) enters the cells but does not translocate to the nucleus, indicating that the nuclear membrane remained intact following digitonin treatment. (B) FITC-rH ferritin enters the cells but is not imported into the nucleus of cells plated in standard culture conditions. (C) If cells are pretreated with deferoxamine and then returned to standard medium containing FITC rH-ferritin, the labeled ferritin translocates to the nucleus. (D) The combination of DFO and ferric ammonium citrate (FAC) results in the formation of ferrioxamine B that limits the chelating ability of DFO. Under this condition, FITC rH-ferritin does not translocate to the nucleus but remains cytoplasmic. (E) FITC-labeled BSA was added to the standard media following DFO pretreatment (same condition as in B) as a control for the selectivity of the uptake mechanism and to show the nuclear membrane is still intact following permeabilization. FITC-BSA enters the cell but does not enter the nucleus. (F) The 222 mutant, an H-ferritin that lacks a ferroxidase center enters the cell nuclei (two cells are shown in this micrograph, one is indicated by the arrow) of DFO-treated cells indicating the iron status of ferritin is not a factor in the nuclear uptake. (G) To determine if there is a preferred subunit type of ferritin that is translocated to the nucleus, FITC rL-ferritin was added to the medium after pretreatment with DFO. This subunit of ferritin does not translocate to the nucleus under these conditions, indicating there is a preference for H-rich ferritin and also demonstrating the integrity of the nuclear membrane after digitonin permeabilization. (A-G) Exogenously applied ferritin. In H, an example of a cell transfected with Myc-tagged ferritin is shown. The epitope tagged ferritin is found in the nucleus of SW1088 cells. These data illustrate that endogenously expressed tagged H-ferritin can translocate to the nucleus. Bars, 7 µm.

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