FIGURE 5.

VASP does not impact MTOC Polarization to the NK cell Cytotoxic Synapse (CS). NKL and primary NK cells were nucleofected with control siRNA (siCtrl) or one of two siRNAs targeting VASP. Seventy-two hours after nucleofection, NKL / primary NK cells were incubated at 37°C to allow conjugation with CMAC-stained 721.221 cells (blue). Cells were fixed and imaged for the location of the MTOC (γ-Tubulin, white) and the location of the lytic granules (perforin, red). The periphery of the cells, as defined by F-actin staining, and is designated by a dotted line. All measurements were measured as the distance between the γ-tubulin location within the NKL / primary NK cell, and the nearest point of synapse with the CMAC stain. (A) Representative example of NKL conjugates allowed to form for thirty minutes and (B, C) quantified for three independent experiments of thirty to fifty conjugates per group per experiment. (D) Representative example of Primary NK cell conjugates allowed to form for thirty minutes and (E, F) quantified for three independent experiments of thirty to fifty conjugates per group per experiment. Error bars indicate SEM. *p < 0.05, ** p < 0.005, and ***p < 0.0005 compared with control group.