Fig. 5. Mechanism of nuclear translocation for FITC labeled rH-ferritin. SW1088 cells were pretreated with deferoxamine for 72 hours followed by digitonin treatment. The cells were then exposed to treatments that affect nuclear translocation of proteins in the presence of FITC-conjugated H-ferritin. The cells in the micrographs are representative of the general population. The arrows indicate the nuclei of individual cells. Each experiment was performed in triplicate. (A) As previously established, FITC-labeled rH-ferritin translocates to the nucleus. (B) Treatment of the cells with wheat germ agglutinin (WGA), to block the nuclear pore complex, blocks nuclear translocation of FITC-labeled recombinant H-ferritin, which collects at the perinuclear membrane. (C) Incubation of cells with FITC-labeled recombinant H-ferritin at 4°C decreased the appearance of the ferritin in cell nuclei. FITC-labeled rH-ferritin collects at the perinuclear membrane. (D) Depletion of ATP in the cells with apyrase results in a decreased appearance of FITC-labeled recombinant H-ferritin in cell nuclei. FITC-labeled rH-ferritin collects at the perinuclear membrane. (E) Regeneration of ATP following apyrase treatment results in FITC-labeled rH-ferritin nuclear translocation. (F) Exposure of the cells to NEM does not inhibit ferritin nuclear translocation. Two cells are shown in this micrograph with immunolabeled nuclei (arrows). These studies demonstrate that ferritin uptake into the nucleus is via the nuclear pore complex; it requires energy but does not require an NLS bearing cytosolic protein. Bars, 7 µm (A-E); 4 µm (F).