Effects of LAMP1 silencing on recruitment of perforin to the immunologic synapse. (A) YTS cells, untransduced or transduced with control (CTRL) or LAMP1 RNAi, were activated by mixing with 721.221 target cells for 30 minutes at 37°C. The cells were next fixed and stained with Ab’s against LAMP1 (red), pericentrin (MTOC marker; blue), and perforin (green). The dashed line indicates the position of the immunologic synapse. Scale bars represent 5 μm. (B) The percentages of perforin polarization to the immunologic synapse in YTS cells conjugated with 721.221 target cells, as in (A). Error bars represent SD. The values were determined by evaluation of 150 conjugates for each indicated YTS cell group in 3 experiments. The diagram (right) illustrates the scoring model. Perforin was regarded as polarized if it localized to a conical area (dark gray region in the diagram labeled as polarization area), limited by the center of the cell and the edge points of the cell-cell interface (shown as the dashed line in the illustration), and the MTOC (as determined by pericentrin staining) was adjacent to the cell-cell contact site.