Rapid delivery of granzyme B and initiation of cell death. Minimal cleavage of Bid by Granzyme B is sufficient for transmission of the cell death signal. (A-B) Granzyme B activity quenching: Jurkat cells were simultaneously treated with perforin and granzyme B (62.5 nM) before shifting cell aliquots to media containing 10 µM compound 20 at 1, 2, 3, 4, 5, 10, or 15 minutes after perforin and granzyme addition. Control treatments included nontreated cells (Ctl) and granzyme B–only (Grz) or perforin-only (Prf) treatments. Cells were then either (B) incubated for a further 10 minutes at 37°C, whole cell lysates were prepared, and the formation of tBid was assessed by Bid immunoblot analysis (top, short exposure; bottom, long exposure) or (A) incubated for 24 hours at 37°C/5% CO₂ and cell viability was assessed. Data represent the mean ± standard error (n = 3 independent experiments). *P < .05, one-way ANOVA, Tukey post hoc test by comparison with no compound 20 (−C20) treatment; ^#P < .05, one-way ANOVA, Tukey post hoc test by comparison with perforin-only (Prf) treatment (all treatments from 2 minutes onward were significantly different to Prf). See also supplemental Figure 2.