Fig 1: High G9 in tumors affects T-cell cytolytic activities. A Mono-(1) Poly- (>1) functional profile of gated CD8 response in G9-knockdown or overexpressed cocultures Pie charts representing the mono-poly-functional profile of gated CD8 response (left) in G9-knockdown or overexpressed cocultures. Size of each pie segment corresponds to the frequency of the corresponding cytokines (color-coded). Arcs depict cytokine makeup within pie slice. Bar graph (right) indicating the percentage of monofunctional profile studied (pie slice in light blue). Representative granzyme B (GzB) staining on cytospun cocultured CTL shown at the side. B Representative zebra plot (left) and quantitative plot (right) of CD107+CD8+ and GzB+CD107+CD8+ cells, respectively. C Schematic representation of the workflow for the co-culture system using an anti-EpCAM antibody to distinguish CD8 + T cells. Sorted cells were then cytospun on the side for GzB staining. Pie chart of GzB percentages with the corresponding representative fluorescence staining images. Scale bar: 20 μm. D Representative staining CD8 (turquoise), granzyme B (GzB; pink), and nuclei (DAPI, blue) in the tumor region of NPC tissues presented with PanCK+G9+ (n = 3) and PanCK+G9− (n = 3). The illustration depicts the distance of a cell (PanCK+G9+ and PanCK+G9−) to another cell type (CD8+GzB + ), defined as the distance between the reference cell (RC) and its nearest neighbor cell (NC) of another cell type. Histogram lines represent CD8+GzB+ paired with PanCK+G9+ cells within 300 µm, with shading indicating an effective distance of 15 µm. The percentage of NC at specified distances relative to RC is shown on the right. E A schematic showing palmitoylation modification allows G9 to translocate onto the cell surface (left column). Representative confocal fluorescence images of E-cadherin (red) and G9 (green) showing cells with (NPC43OE6-Palm; bottom) and without (NPC43OE6; middle) palmitoylation. NPC43Vector as control. The middle column, line profiles quantifying fluorescence signals from E-cadherin (red) and G9 (green) along the yellow lines in the representative image. Scale bar: 10 μm. The right column, sorted cells were then cytospun on the side for GzB staining with corresponding pie chart of GzB percentages. Scale bar: 5 μm. F Schematic illustration of tumor cells loaded with exogenous, activated GzB protein (10 μg) with pore-forming protein streptolysin-O. Endogenous GB puncta Green masks and magnified insets of representative regions staining (left) and quantitative data on GzB puncta in target NPC cells. Scale bar: 50 μm. G Schematic representation of GranToxiLux assay. Representative flow cytometry analysis of granzyme B activity (FITC + ) into the TFL4+ TarTC upon cocultures (left) and quantitative data on percentage of death TarTC (GzB-incorporated target cells) (right). The data were pooled from three (n = 3) independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 (one-way ANOVA test for (A–C), (E–G); two-tailed unpaired Student’s t-test for (D)). Data points are shown ± SEM