Effect of trastuzumab and T-DM1 on cell death and antibody dependent cell cytotoxicity. (A) Dead cells were counted after 24, 48 and 72 h of exposure to trastuzumab or T-DM1 (1 μg/ml) and the percentage of dead cells was calculated. (**p < 0.01, ***p < 0.001 versus control, one-way ANOVA followed by Tukey’s post-test). (B) Caspases 7 and 9 activation were detected by immunoblotting on cell lysates obtained after 48 h of Calu-3 exposure to increasing concentration of trastuzumab or T-DM1. Vinorelbine 0.001 μg/ml was used as positive control. (C) Cytochrome c was detected in the cytoplasm by immunoblotting after 48 h of treatment with T-DM1 1 μg/ml as described in Methods section. (D) Trastuzumab (1 μg/ml) or T-DM1 (1 μg/ml) were added to Calu-3 and H1299 cells seeded with 100 U/ml IL-2 activated-NK cells, at the ratio of 1:50. After 4 h lactate dehydrogenase release was quantified as described in Methods section and data expressed as percentage of cytotoxicity. The results are from representative experiments. The experiment, repeated three times, yielded similar results (***P < 0.001, one-way ANOVA followed by Tukey’s post-test).