Fig 2: Characterization of cortactin expression and subcellular localization in PDAC cell lines and establishment of cortactin knockdown mediated by CRISPR/Cas9 technology. a Immunoblotting of whole cell lysates of PDAC cell lines for cortactin and Src and their activated forms (p-cortactin Tyr421 and p-Src Tyr416). The MCF-7 breast carcinoma cell line was included as positive control. b Cortactin localization (green) in BxPC-3 and Panc-1. The nucleus was counterstained with DAPI (blue). Arrowheads indicate cortactin expression at sites of cell–cell-contacts. Scale bar, 50 µm. c Serum starved Panc-1 cells were stimulated with 10% FCS for 1 h. After stimulation, cortactin (green) localized to the cell membrane. The actin cytoskeleton was stained with phalloidin (red) and the nucleus was counterstained with DAPI (blue). Scale bar, 50 µm. d Immunoblotting of CRISPR/Cas9 mediated cortactin knockdown (CTTN KO) cells. Cells were grown as single selected clones. GAPDH was used as loading control

Fig 3: Functional analysis of cortactin knockdown (CTTN KO) cells and controls (CTRL). a, c Knockdown of cortactin did not affect pancreatic cell proliferation measured with the xCELLigence system of BxPC-3 and Panc-1 cells. b, d Cell migration assessed in vitro performing scratch assays showed significantly impaired migration of CTTN KO cells compared to the controls. e Cell migration stimulated with 0–2.5% FCS was measured with the xCELLigence system and showed significantly reduced migration in Panc-1 CTTN KO cells. Bar chart indicates the relative cell migration normalized to control cells after 8 h. f Quantification of matrix degradation. No significant differences were observed between BxPC-3 CTTN KO cells and controls. g BxPC-3 cells were stained with DiO prior to spheroid culture for collagen cell invasion assays. Spheroids cultured for 24 h were then embedded in a collagen suspension and cell invasion was assessed 12 and 24 h after plating. BxPC-3 CTTN KO cells showed significantly impaired invasion compared to the controls. Scale bar, 1000 µm. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant. Data points shown are the mean + SD. The results are representative of at least three independent experiments