Fig. 4

Src inhibitor dasatinib mediated effects in pancreatic cancer cell lines adherently cultured or as spheroids. a Dasatinib treated adherently cultured BxPC-3 cells showed decreased Src Tyr416 and cortactin Tyr421-phosphorylation levels compared to control cells. b Immunofluorescence analysis of (I) cortactin or (II) p-cortactin Tyr421 after dasatinib treatment. Green fluorescence indicates (I) cortactin or (II) p-cortactin staining, the actin cytoskeleton is shown in red and the nucleus is shown in blue. b I Morphological changes (epithelial to mesenchymal) after dasatinib treatment (1 µM) were detected in Panc-1 cells. Scale bar, 20 µm. b II Immunofluorescence for p-cortactin Tyr421 revealed a delocalization of phosphorylated cortactin from the cell membrane to the cytoplasm after dasatinib treatment. Scale bar, 20 µm. c In scratch assays, BxPC-3 and Panc-1 cells treated with 50 nM dasatinib or DMSO as control showed a significant decrease in cell migration ***p < 0.001. d Increased cortactin expression and Tyr421 phosphorylation levels after 6 days of spheroid culture compared to adherently cultured cells were detected in pancreatic cancer cell lines BxPC-3, Capan-1 and Panc-1. adh, adherent; sph, spheroid. e Elevated Tyr421-phosphorylation of cortactin and higher expression of E-cadherin at cell–cell contacts of agarose embedded BxPC-3 cells in spheroid cultured cells (6 days) were determined by immunohistochemistry. Scale bar, 100 µm. f Immunoblotting of BxPC-3 and Panc-1 cells cultured in spheroids and treated with dasatinib (BxPC-3: 100 nM; Panc-1: 1 µM) for 24 h showed less induction of E-cadherin and cortactin expression compared to control cells. Immunoblotting of p-ERK 1/2 (Thr202/Tyr204) and ERK kinase were included to verify dasatinib specificity. GAPDH was used as loading control