Internalization of soluble forms of recombinant tumor necrosis factor-related apoptosis-inducing ligand:iron superoxide dismutase (sTRAIL:FeSOD) is rapid and is inhibited by hyperosmotic sucrose. (A) through (C) Suspended erythroleukemia (K562) cells were treated with fluorescein isothiocyanate (FITC)-labeled sTRAIL:FeSOD (green) in RPMI 1640 medium without fetal calf serum for (A) 5 minutes, (B) 15 minutes or (C) 30 minutes. After acid washes, cells were visualized with laser scanning confocal microscopy (LSCM). The LSCM parameters were set to an excitation wavelength of 488 nm, an emission wavelength of 500 to 550 nm and pinhole of 202 μm. (D) LO2 cells were preincubated for 30 minutes in the presence of 0.25 M sucrose at 37°C before incubation in the presence of FITC-labeled sTRAIL:FeSOD (green) for an additional 30 minutes. After being washed extensively in ice-cold phosphate-buffered saline (PBS), cells were counterstained with Hoechst 33342 (blue) and analyzed by LSCM. The white bar represents 5 μm. The LSCM parameters for FITC were set to an excitation wavelength of 488 nm, an emission wavelength of 500 to 550 nm and pinhole of 215 μm. For Hoechst 33342, the parameters were set to an excitation wavelength of 350 nm, an emission wavelength of 460 to 480 nm and pinhole of 215 μm. (E-H) After K562 cells were treated with PBS, FITC-labeled FeSOD, sTRAIL:FeSOD or sTRAIL:mFeSOD for 30 minutes, the internalization rate was quantified by flow cytometry.