DFP-mediated mobilization of iron from H9C2 cells to extracellular apotransferrin: receptor-mediated uptake of holotransferrin as an index of iron transfer. H9C2 cells were pretreated with succinylacetone as described in “Methods” to increase mitochondrial labile iron levels. They were then incubated at 37°C in DMEM-HEPES medium containing 20 μM lissamine R-aTf with various additions, and epifluorescence microscopy images were obtained under rhodamine settings after 60 minutes of incubation. Representative images of cells with no addition (A), 30 μM DFP (B), 100 μM unlabeled holotransferrin (C), 100 μM unlabeled holotransferrin with 30 μM DFP (D), 20 μM Fe-NTA, 1:3 ratio (E), and 20 μM Fe-NTA with 30 μM DFP (F). (G) The illustration represents entry of DFP into cells, mobilization of iron from the cytosol (C), nuclei (N) and mitochondria (M), followed by exit of DFP-Fe complexes from the cells (step 1). This is followed by transfer of iron from DFP-Fe to R*-aTf to form R*-Tf-Fe (step 2), which then binds to transferrin receptors on the cell surface (step 3), concentrates in the endosomes, and is detected by fluorescence microscopy as punctuate fluorescence typical of microvesicles. (H) Mean cell-associated fluorescence values in r.u. of 5 cells per field (± SD, from 3 separate experiments), calculated from snapshots such as shown in panels A through F, obtained after 1 hour of incubation.