Blocking GzmB in target cells overexpressing Bcl-2 and XIAP causes increase in synapse dwell time and cytokine production. (A) Western blot analysis of unmodified, Bcl-2–overexpressing, or both Bcl-2– and XIAP-overexpressing HeLa cells. (B) Cytotoxicity assay of human NK cells co-cultured with ⁵¹Cr-labeled HeLa cells (parental or overexpressing Bcl-2 and XIAP) in the presence or absence of the human GzmB inhibitor compound 20 (C20). Shown is the percentage of specific ⁵¹Cr release at E/T of 10:1 of three independent pooled experiments (mean ± SEM). Statistical significance (*, P < 0.05) is shown. (C) Fluo-4–AM-labeled human NK cells were added to HeLa cells or HeLa Bcl-2^hiXIAP^hi cells in the presence of C20 to inhibit GzmB during time-lapse microscopy. After conjugate formation, synapse time was measured from the first Ca²⁺ flux in NK cell until it detached from the target cell and is shown as synapse dwell time (mean minutes ± SEM; n = 18 and 15, respectively). Each data point represents one conjugate; data points filled with yellow indicate conjugates, where NK cells were still attached to the targets at the end of the video, underrepresenting the attachment time. The supernatant from these experiments were then analyzed for cytokine secretion by CBA, and shown is the concentration of cytokines produced after 5 h (mean pg/ml ± SD of representative experiment of two). Statistical significance was determined by Tukey’s post-hoc ANOVA. *, P < 0.05.