FIGURE 4.

VASP does not impact NK cell conjugate formation. NKL and primary NK cells were nucleofected with control or two siRNAs targeting VASP. (A-F) Seventy two hours after nucleofection, NKL and primary NK cells were allowed to form conjugates with CMAC-stained 721.221 cells (blue) at 37° and imaged for the localization of LFA-1 or talin, as indicated. (B, D, F) The amount of synaptic LFA-1 or talin was quantified in relation to the amount of cellular protein and the size of the synapse, as described in the methods. Results were quantified for thirty to fifty conjugates per group for each of three independent experiments for each finding. (G, H) 72 hours after nucleofection, NKL and primary NK cells stained with Calcein-AM and then allowed to form conjugates at 37°C with CMAC-labeled 721.221 cells over the specified times and then fixed. The formation of conjugates (CMAC + Calcein-AM+) in respect to the total number of NK / NKL cells (Calcein-AM+) was assessed using two color flow cytometry. Results shown are the average of 5 (primary NK) or 7 (NKL) replicate experiments, each performed in triplicate. Error bars indicate SEM. *p < 0.05, ** p < 0.005, and ***p < 0.0005 compared with control group.