Fig. 5

TGIF1 loss promotes EMT and CSC activity to increase PDAC cell migration and invasiveness. a Morphological characterization of primary PDAC cells derived from PKP and PKTP mice through phase-contrast microscopy. Scale bar =100 μm. b Immunocytochemical analysis of epithelial markers, CK19 or E-cadherin, in PKP and PKTP PDAC cells. Primary cell cultures of PDAC cells were stained for CK19 and E-cadherin. Scale bar =100 μm. c Western blot analysis by using the total cell lysate of murine PDAC cells derived from PKP and PKTP mice revealed a decrease in E-cadherin, integrin β1 expression, TGIF1 expression, and an increase in SMA, N-cadherin, Vimentin, and Zeb1 expression in PKTP cells as compared to PKP cells. d TGIF1 loss has no significant effect on PDAC cell proliferation in vitro. Cell proliferation was determined using the MTT assay. Data are represented as mean ± s.d; N = 3 independent experiments. e&f TGIF1 significantly inhibits the in vitro tumor sphere-forming ability of PDAC cells, whereas PKTP cells enhanced the anchorage-independent growth e and tumor sphere formation in “sphere”-forming assays f, compared with PKP cells. g i, Engrafted PDAC formation of PKTP and PKP PDAC cells in a SCID mouse xenograft model. ii, Average tumor weights and volumes/sizes of the PKTP groups significantly increased compared with the control PKP groups ***p < 0.001.. h, Wound healing assays showed that TGIF1 loss stimulates in vitro cell motility of murine PDAC cells. i Transwell invasion assays demonstrated that TGIF1 gene ablation enhances the invasive ability of murine PDAC cells. Representative images are from three independent experiments. j Western blot analysis of the total and phosphorylated Akt, Erk (p-44/42), STAT3, p38 MAP kinase pathways and cancer stemness marker Nanog, Sox2, Nestin, CD44, and CD133 in PKP and PKTP PDAC cell lines. β-actin served as a loading control. k Detection of the stem-cell-specific markers CD44, CD133 and Nanog in PKP and PKTP PDAC cells as determined using immunocytochemical analysis. Scale bar = 100 μm