Fig 2: The PRMT5-E2F1 axis controls cell motility.a Migration assay performed in p53-/- WT E2F1 and p53-/- E2F1 Cr HCT116 cells after 2 days of treatment with 1 µM T1–44 or DMSO control. The rate of migration was determined by analysing the slope of the line between 4 and 10 h intervals, and is presented in the bar charts to the right. n = 3. b An invasion assay was performed under conditions identical to those described above. The rate of invasion was determined by analysing the slope of the line between 4 and 8 h intervals, and is presented in the bar charts to the right. n = 3. c An adhesion assay was performed under conditions identical to those described above. The rate of adhesion was determined by analysing the slope of the line between 0 and 0.4 h intervals, and is presented in the bar charts to the right. n = 3. d An immunoblot demonstrating input protein levels for E2F1, p53 and SDMe for the experiments in a, b, and c is displayed; actin included as a loading control. e Adhesion assay performed in p53-/- WT E2F1 and p53-/- E2F1 Cr HCT116 cells in the presence of CTTN siRNA. The rate of adhesion was determined by analysing the slope of the line between 0 and 0.5 h intervals, and is presented in the bar charts to the right. n = 3. f Immunoblot displaying input protein levels for CTTN, E2F1 and p53 for the experiment in e is presented; actin included as a 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

Fig 4: Gelsolin is a novel interaction partner of cortactin in PDAC. a, b Verification of mass spectrometry results by Co-immunoprecipitation (Co-IP) of gelsolin by cortactin antibody. Co-IP with cortactin antibody showed cortactin precipitation and co-precipitation of gelsolin in a BxPC-3 and b Panc-1 cells. CTTN KO cells served as negative controls. Black arrows indicate size of gelsolin and cortactin. c Immunofluorescence staining of gelsolin (green) and cortactin (red) confirmed the possible interaction as co-localization of both proteins could be detected. Scale bar, 50 µm. d In cells cultured in spheroids upregulation of gelsolin expression compared to cells in adherent culture was detected. GAPDH was used as loading control. e Scheme of the role of cortactin in pancreatic cancer migration. ab, antibody; adh, adherent; IB, immunoblot; IP, Immunoprecipitation; sph, spheroid; WCL, whole cell lysate; Wt, wild type

Fig 5: Cortactin/CTTN regulates motility through the PRMT5-E2F1 axis.a Migration assay performed in WT E2F1 p53-/- HCT116 cells in the presence or absence of CTTN siRNA, after 2 days of treatment with 1 µM T1–44 or DMSO control. The rate of migration was determined by analysing the slope of the line between 4 and 10 h intervals, and is presented in the bar charts to the right. n = 3. b An invasion assay was performed under identical conditions as described above. The rate of invasion was determined by analysing the slope of the line between 4 and 8 h intervals, and is presented in the bar charts to the right n = 3. c An adhesion assay was performed under identical conditions as described above. The rate of adhesion was determined by analysing the slope of the line between 0 and 0.4 h intervals, and is presented in the bar charts to the right. n = 3. d An immunoblot demonstrating input protein levels for CTTN, p53 and SDMe for the experiments in a, b and c is displayed; actin included as a loading control.