Perforin and Ad2 cause an intracellular redistribution of granzyme B. (A) Jurkat cells were pretreated with granzyme B at 37°C for 60 minutes to allow uptake of granzyme. Cells were washed and incubated in either perforin or Ad2. Cells were immunolabeled for granzyme B and Texas Red (TR)-conjugated secondary antibody (red) and TUNEL labeled with dUTP-FITC (green). Regions of colocalization appear yellow. The punctate cytoplasmic labeling pattern of granzyme B alone (see Fig 1 A) was disrupted and nuclear accumulation of granzyme B was evident. The presence of granzyme B in the nucleus coincided with sites of DNA fragmentation detected by the TUNEL protocol. (B) COS M5 cells were transiently transfected to express granzyme B. After 48 hours, cells were treated with buffer as a negative control or perforin and labeled for granzyme B (TR) and TUNEL (dUTP-FITC) as in (A). In the absence of perforin, granzyme B appeared to be contained in a cytoplasmic structure, but was redistributed in the presence of perforin. As with Jurkat targets, COS cells containing granzyme B underwent rapid apoptosis when treated with perforin and nuclear granzyme B was colocalized with sites of TUNEL-labeled DNA fragmentation. Scale bar is 10 μm.