Phosphatidylserine externalization and nuclear condensation occur in GzmC-induced apoptosis.
(A) Percentage of target cells with single-positive annexin Vhi (AV) versus double-positive AVhi/7-AADhi staining. YAC1 target cells were treated with perforin and 1μM rGzmB or rGzmC at 37°C for 5, 15, 30, or 60 minutes. Additionally, target cells were exposed to nothing, perforin, rGzmB, or rGzmC alone. Results are representative of 2 independent experiments. (B) YAC1 cells were treated with perforin alone or perforin plus 1 μM rGzmB or rGzmC for 15 minutes at 37°C. Cell suspensions were immobilized onto microscope slides, stained by Wright-Giemsa, and visualized by light microscopy (× 100 magnification). Cells treated with perforin showed a normal morphologic appearance. Target cells that were untreated or treated with granzymes only resembled perforin-treated cells (data not shown). Nuclear collapse (arrows) was observed, as was late cell disruption (arrowheads). (C) Transmission electron microscopy of YAC1 cells incubated with perforin and granzymes as in panel B revealed chromatin condensation (arrows) and cytoplasmic disruption (bar represents 2.5 μm). Perforin-treated cells were identical to untreated cells or to cells treated with granzymes only (data not shown).
