Figure 2.

Rapid cell entry and nuclear targeting of PCI. (A) Internalization of PCI into live cells. Leukocytic HL-60 cells were incubated for 10 min with 90 nM of biotin-labeled uPCI (green) at 37°C, and intracellular staining of PCI was monitored by confocal microscopy at a focal plane allowing visualization of nuclei. Cell nuclei were stained with TO-PRO-3 (red). The images are representative of five independent experiments. (B) Intracellular PCI is directed to intranuclear compartments. Immunoelectron micrographs of HL-60 cells incubated with 90 nM of biotin-labeled rPCI for 0 (control, immediately processed for the analyses) and 30 min at 37°C. Arrows indicate PCI detected by gold-labeled anti-biotin antibody. No nuclear labeling of PCI is seen under control conditions. After 30 min of incubation, the serpin accumulates in the nucleus (N). Bars, 0.5 μm. (C) PCI is targeted to leukocyte nuclei in vivo. Biotin-labeled uPCI injected into the murine blood circulation is recovered within 30 min in the nuclei of granulocytes. Colabeling of biotin-PCI with TO-PRO-3 in live granulocytes documents the nuclear localization of the serpin.