Activation of STAT3 by S727E directs loss of epithelial identity and partial EMT. (A) Western blot analysis of STAT3 Y705 and S727 phosphorylation in nuclear and cytoplasmic lysates from KRASG12D p53 knockout pancreatic cells maintained in serum-free 2D, 3D Matrigel (3DM), and 3D suspension (3DS) cultures. (B) FACS analysis of CD133, EpCAM, and SCA1 expression in KRASG12D p53 knockout pancreatic cells and their STAT3 derivatives. (C) IHC (KRT19 and pY705 STAT3) staining of mixed tumors arising from KRASG12D pancreatic cells expressing STAT3 S727E and the tyrosine phosphorylation-deficient STAT3 Y705F/S727E double mutant. (D) Representative images of zebrafish xenografts containing KRASG12D cells or their STAT3 derivatives at 4 d after injection. Red fluorescence indicates zebrafish blood vessels, and green fluorescence is transplanted mouse cells. Arrowheads indicate cells with various invasion capabilities. (E) Metastatic tumor formation in the lungs of nude mice injected by tail vein with KRASG12D p53 knockout pancreatic cells and their STAT3 derivatives. n = 4 for each cell type. Values correspond to average and SD. Asterisks indicate a statistically significant difference compared with the STAT Y640F group. (F) H&E staining of metastatic tumors in the lungs of nude mice injected with KRASG12D p53 knockout cells and their STAT3 derivatives (shown in E).