Figure 6. EpCAM promotes β-catenin expression and CTL persistence.

(A) EpCAM expression was measured in OT1 CTLs transduced with EpCAM or control (Ctrl) retrovirus. (B) A 1:1 mixture of EpCAM and Ctrl CTLs was transferred into B16OVA tumor bearing mice. After 1 week, CTLs were extracted from various organs and quantified by flow cytometry. n ≥ 3 for each group ***P ≤ 0.001 and ****P ≤ 0.0001, calculated by two-way ANOVA. (C) β-catenin (βcat) in CTLs expressing EpCAM or empty vector (Ctrl), assessed by immunoblot using actin as a loading control. (D and E) OT1 CTLs expressing the indicated miRs were fixed and stained for β-catenin together with DAPI to visualize the nucleus. (D) representative z-projection images (scale bars = 5 μm). (E) quantification of mean nuclear β-catenin intensity. n ≥ 13 cells for each group. (F and G) Intracellular localization of GFP (F) and EpCAM-GFP (G) in OT1 CTLs is shown. Representative images of GFP fluorescence and nuclear DAPI staining are shown to the left (scale bars = 7 μm). Normalized intensity linescans, derived from the yellow lines in the images, are shown to the right. (H and I) CTLs expressing EpCAM-GFP or GFP alone were subjected to PLA using antibodies against GFP and β-catenin. (H) Representative images of PLA signal together with nuclear DAPI staining (scale bars = 7 μm). To the left of each image, schematic diagrams illustrate predicted PLA results assuming a specific interaction between EpCAM and β-catenin. (I) Nuclear PLA puncta in EpCAM-GFP (EpC) and GFP transduced CTLs were quantified. n ≥ 26 cells for each group. All error bars indicate SEM. P-values in (E) and (I) were calculated by Student’s t test. All data are representative of at least 2 independent experiments.