GzmC induces target cell mitochondrial depolarization, swelling, and cyt

c release in intact cells. (A) High-magnification transmission electron microscopy (× 22 000). The arrows point to the mitochondria of YAC1 cells treated as described in Figure 2C (bar represents 0.4 μm). The mitochondria of untreated YAC1 cells or of cells incubated with granzymes alone has the same appearance as perforin-treated mitochondria (data not shown). (B) Representative pseudocolor-coded images of TMRM fluorescence intensity in murine embryonic fibroblasts at the beginning (left panels) and at the end of the acquisition sequence (right panels). Cells were treated with perforin alone or with perforin plus 4 μM GzmC. Where indicated, cells were preincubated with 2 μM CsA for 30 minutes. Bar represents 7 μm. (C) Quantitation of the TMRM fluorescence changes over mitochondrial regions. Where indicated by the arrows, perforin and 4 μM rGzmC, and then 2 μM FCCP (to induce complete depolarization), were added. Fluorescence intensity changes were quantified as described in “Experimental procedures.” (D) Immunofluorescence for cytc in YAC1 cells. YAC1 target cells were treated with perforin alone (i-ii), perforin plus 1 μM rGzmB (iii-iv), or perforin plus 1 μM rGzmC (v-vi) for 30 minutes and were immunostained for cytc (red). Duplicate images in the lower panels (ii,iv,vi) show nuclear staining by DAPI (blue). Note that target cells treated with perforin plus granzymes display diminished cyt cstaining and have apoptotic condensed nuclei. Untreated target cells and cells treated with granzymes alone resembled those treated with perforin only (i,ii, and data not shown).