Septin filament dynamics and septin filament formation are required for NK cell cytotoxicity. (A and C) DMSO- (control) or FCF-treated cells were incubated at 37°C for 3 h with ⁵¹Cr-labeled 721.221 cells at the indicated E:T ratios, and specific lysis was calculated. (B and D) Results were quantified for an E:T ratio of five effectors to one target cell over six independent experiments. (E–I) NKL cells were nucleofected with siCtrl or siRNAs targeting septin 2. Immunoblot is cropped for clarity. (E) 72 h after nucleofection, levels of septin 2 protein were assessed by immunoblot for each experiment. (F) Cytolytic activity of nucleofected cells against ⁵¹Cr-labeled 721.221 cells was measured as indicated above. (G) Results were quantified for an E:T ratio of 1.25 effectors to one target cell over four independent experiments. (H) Nucleofected NKL cells were incubated with ⁵¹Cr-labeled p815 cells coated with anti-NKG2D + anti-2B4. Percent specific lysis was calculated. (I) Results were quantified at an E:T ratio of 1.25 over four independent experiments. All experiments were completed in triplicate or quadruplicate and in reference to spontaneous and total lysis. Results show representative experiments (A, C, E, F, and H) or average values over multiple experiments (B, D, G, and I). Error bars indicate SEM. **, P < 0.005; ***, P < 0.0005 compared with control group. P values indicated are for paired, two-tailed Student’s t test.