Fig. 1: Activation of EphA2 receptors causes rearrangement of the cytoskeleton, retraction of cell protrusions and cell rounding.

a Western blot analysis showing EphA2 receptor phosphorylation and inhibition of AKT, MEK/ERK and FAK/SRC signaling after stimulation with 1 µg/ml clustered ephrin-A1-Fc (efnA1-Fc) for up to 3 h, as indicated, in PC-3 prostate cancer cells. The full 72-h time course is shown in Supplementary Fig. 1d. Confocal microscopy images of the F-actin cytoskeleton labeled with rhodamine-phalloidin (red) in PC-3 cells in response to stimulation with clustered efnA1-Fc compared to Fc control in the absence (b) and the presence (c) of Cytochalasin D (CytoD). Cell nuclei were labeled with DAPI (blue). Incubation times with clustered ephrin-A1-Fc and Fc control, respectively, are indicated in the figure. Cell outlines, based on corresponding brightfield images, are indicated as white lines in the CytoD-treated cells. d Confocal microscopy images of microtubules (α-tubulin, green) in unstimulated and PC-3 cells stimulated for 2 min with clustered efnA1-Fc. Cell outlines and nuclei are visualized using F-actin staining (rhodamine-phalloidin, red) and DAPI (blue), respectively. Anisotropy of microtubules was quantified using the ImageJ plugin FibrilTool. Data represent means ± SE (n > 40 individually analyzed cells). ***p < 0.001 (unpaired t-test) e Quantification of PC-3 cell adhesion to uncoated, fibronectin-, Matrigel-, clustered Fc-control- or efnA1-Fc-coated cell culture surfaces after 30 min. Data represent means ± SE (n = 4 independent experiments). *p < 0.05, ***p < 0.001, ns. not significant (repeated-measure ANOVA with post-hoc Tukey’s multiple comparisons tests). f Fluorescent microscopy images of PC-3 cell adhesion to glass coverslips coated in alternating stripes of clustered efnA1-Fc (10 µg/ml) and either clustered Fc control (10 µg/ml) or Matrigel (1:100) as an alternative substrate for adhesion. Cells were fixed at 1 h and 24 h after plating, then visualized with F-actin (rhodamine-phalloidin, red) and DAPI (blue) labeling. g, h Time-lapse brightfield microscopy images of PC-3 cell invasion into Matrigel containing either 1 ug/ml Fc control or clustered efnA1-Fc. Initial cell-to-Matrigel boundary at t = 0 is indicated as a black line. Colored asterisks and arrow heads follow individual cells and cell protrusions, respectively, over time.