Kinesin-1 controls the terminal transport of lytic granules after granule polarization. (A) Top panel: confocal microscopy of CTLs transfected with GFP-KIF5B. Cells were fixed, permeabilized, and stained with anti-tubulin (red). Bottom panel: confocal microscopy of CTLs transfected with GFP-KIF5 DN and stained with anti-tubulin (red). The insets represent an enlarged image. Scale bars represent 5 μm. All images of single cell were representative of > 60 cells observed over at least 4 independent experiments performed in duplicate. (B) Lak cells were transfected with CFP-KIF5B or CFP-KIF5 DN. Transfected cells were incubated for 3 hours alone or with a coated anti-CD3 antibody in the presence of anti-CD107a–FITC and anti-CD107b–FITC antibodies. Thereafter, cells were stained with anti-CD3–PE and anti-CD8–APC antibodies. Lymphocytes were gated on CD3⁺CD8⁺ cells. Profile shows CFP versus CD107-FITC gating on CD3⁺CD8⁺ CTLs. For each condition, a histogram overlay analysis of Lamp secretion by CFP⁺ cells was performed. Each condition is representative of 3 independent experiments performed in duplicate. (C) Confocal microscopy of CTLs transfected with mCherry-KIF5 DN and conjugated with L1210 target cells. Cells were then fixed, permeabilized, and stained with anti-perforin antibody (green). Scale bars represent 5 μm. All images of conjugates were representative of > 90 cells observed over at least 3 independent experiments performed in triplicate. (D) CTLs cotransfected with CFP and siRNA control or siRNA targeting KIF5B were incubated for 3 hours alone or with a coated anti-CD3 antibody in the presence of CD107a and CD107b antibodies. CFP⁺ cells were analyzed for Lamp secretion and shown in a representative histogram of 3 independent experiments. Statistical analysis was performed by Mann-Whitney test. *P < .05.