Fig 2.

Enlarged lytic granules in LYST-deficient NK cells are functional. A, Granzyme B activity in total cell lysates. Granzyme B–negative 721.221 cells served as a control. B, Proteolytic activity of cathepsin B evaluated by using flow cytometry. Concanamycin A–treated cells served as a negative control. Numbers indicate median fluorescence intensity values. Confocal images show visualization of cathepsin activity in the cells; insets indicate differential interference contrast (DIC) images. Scale bars = 5 μm. C, Lysosomal pH. pH values were determined, as described in the Methods section. LYST CRISPR cells treated with concanamycin A were used as a control. D, Lytic granule analysis. The presence of proteins in the total cell lysate (T), purified lysosomal fraction (L), and cytoplasmic fraction (C) was assessed by means of immunoblotting with antibodies specific for the indicated proteins. Each lane was loaded with 1 μg of protein material. E and F, Perforin polarization to the immunologic synapse. Cells were stained with antibodies against perforin (green), Rab27a (blue), and VAMP7 (red; Fig 2, E) or with phalloidin (F-actin marker; blue) and antibodies against pericentrin (MTOC marker; red) and perforin (green; Fig 2, F). The dashed line in Fig 2, E, shows the position of the immunologic synapse. DIC, Differential interference contrast. Scale bars = 5 μm. Graphs in Fig 2, F, show the mean distance between perforin-positive granules and the immunologic synapse or MTOC. Graphs show mean values with SDs. **P < .01, 1-way ANOVA (Fig 2, C) or the unpaired t test (Fig 2, F). ns, Not significant. Fig 2, A, C, and D, data from 3, 4, or 2 experiments, respectively; Fig 2, F, n = 18 conjugates for each cell group.