siRNA-mediated knockdown of CD107a in primary NK cells increases their apoptosis after target cell encounter. (A) Kinetic of CD107a knockdown. Human NK cells transfected with siRNA against CD107a or control siRNA (CTRL) were analyzed at the indicated day by anti-CD107a and anti-actin Western blotting analysis. KD, knockdown efficiency in relation to siCTRL-treated cells normalized to actin. (B) Left: Degranulation on day 2 after siRNA transfection in response to K562 cells. Cells were coincubated at an E/T of 0.5 for 3 hours in the presence of anti-CD107a antibodies and analyzed by FACS. Right: MFI of CD107a within the CD56⁺ gate was determined for siCTRL- (–) and siCD107a- (+) transfected NK cells. Statistics were performed using the paired 2-tailed Student t test; n = 5 ± SD. (C-F) Human NK cells transfected with siRNA against CD107a or control siRNA were incubated for 4 hours in the presence of anti-CD107a antibodies without (E) or with (C,F) K562. Cells were stained for CD56, followed by Annexin-V staining, and then analyzed by FACS. (C) K562 were identified by gating on CD56-negative events (top) and analyzed for Annexin-V staining. Data were plotted as mean ± SEM (right). Statistics were performed using the paired 2-tailed Student t test (C-D). (D) CD56⁺ NK cells were analyzed for Annexin-V staining in the absence or presence of K562 target cells. A representative staining is shown on the left. Statistical summary of data from 5 independent experiments are shown on the right. (E-F) The data shown in (D) were analyzed in relation to CD107a staining. See “Material and methods” for details on normalization. Dotted lines indicate equal ratios.