Ciliobrevin D increases bystander killing of the neighboring cells. Flow cytometry–based cytotoxicity assay of NK cells treated with ciliobrevin D or DMSO control was performed as described in Materials and methods. Raji cells were used as nonsusceptible bystander cells to measure the degree of collateral damage caused by nondirectional degranulation. Specific lysis of the corresponding susceptible targets 721.221 and K562 cells by YTS (A, left) and NK92 (B, left) cells was not affected by ciliobrevin D treatment, whereas the nonspecific lysis of Raji cells by YTS (A, right) and NK92 (B, right) cells increased after ciliobrevin D treatment (100 µM) compared with DMSO control. The cytotoxic function of eNK cells against K562 cells was also not affected (C, left), and the bystander killing of Raji cells was increased (C, right). Data from three independent experiments for YTS and NK92 cells and three healthy donors for eNK cells is shown. Colored dots denote individual experiments or donors. Error bars show ± SD. *, P < 0.05; ****, P < 0.0001; ns, not significant.