Figure 7: Metarrestin specifically binds eEF1A2, and increased eEF1A2 enhances PNCs and metastasis formation.

(A) The structure of metarrestin tagged with biotin. (B) Metarrestin effectively outcompeted recombinant eEFl A from binding to anchored biotinylated metarrestin-eEF1A complex. (C) Metarrestin treatment stabilized eEF1A in a thermal stability assay using PC3M cell lysate. (D) Western blot analyses do not show changes in the amount of eEF1A proteins upon metarrestin treatment at 1 μM for 24 hours. (E) Overexpression of HA-eEF1A2 enhanced PNC structures in cells (image panels, each containing a single nucleus). Although it did not significantly increase overall PNC prevalence (top), overexpression of HA-eEF1A2 increased the number of PNCs per nucleus (scattered PNC prevalence: the number of cells containing 2 or more PNCs); n = 300 cells (bar= 2 μm). (F) Overexpression of eEF1A2 in PC3M cells increased the IC₅₀ of metarrestin for PNC disassembly. (G) 6×10⁴ PANC1 3D spheres transduced with empty vector (control) or eEF1A2 (eEF1A2 O.E.) were injected into the tail of the pancreas of NSG mice. Mice from both groups were harvested 6 weeks after implantation and subjected to necropsy. Macroscopic images of anterior (top) and posterior (bottom) liver surfaces showed higher metastatic burden in PANC eEF1A2 animals than in empty vector control (left, harvested livers). Histopathological images (H&E) of livers (black scale bar=250 μm, white scale bar=100 μm) are shown on the right. Insets depict representative metastatic lesions. Quantification of liver metastasis showed a higher metastatic burden in PANC1 eEF1A2 O.E. animals (n=4 animals analyzed per group). * P < 0.05, ** P < 0.01.