Fig 3.

Rab14 silencing in LYST-deficient NK cells restores the normal size of lytic granules and cytotoxicity. A, Rab14 localization. The indicated NK cells were stained with antibodies against perforin (green) and Rab14 (red). The dashed line shows the position of the immunologic synapse. Scale bars = 5 μm. DIC, Differential interference contrast. B, Rab14 silencing. LYST CRISPR NK92mi cells were mock transduced (M) or transduced with scramble RNAi (S) or 2 Rab14 RNAis (R1 and R2). Protein levels of Rab14 were analyzed by means of immunoblotting; actin served as a loading control. EV, Empty vector CRISPR-transduced cells; UT, untransduced cells. C, LYST CRISPR cells transduced with scrambled (SCR) RNAi or Rab14 RNAi (R1) were stained for perforin (green) and Rab14 (red). Insets show DIC images. Scale bars = 5 μm. D, Frequency distribution of perforin-positive granule sizes; inset shows the average diameter of perforin-positive granules. E, Average number of perforin granules after transduction with the indicated RNAi. Error bars in Fig 3, D and E, indicate SDs. ****P < .0001, unpaired t test. N = 28 (SCR RNAi) or 26 (Rab14 RNAi) cells from 2 experiments. F, Cytotoxicity of the indicated cells at different effector/target (E:T) ratios. Untransduced and EV CRISPR–transduced NK92mi cells served as controls. Graph shows mean values + SDs determined from 3 experiments.