Figure 2: RCP and EphA2 influence cell:cell repulsion.

(a–c) PC3 cells were transfected with siRNAs targeting RCP (SMARTPool (si-RCP) or two individual oligos (si-RCP#1 and si-RCP#2), FIP2 (si-FIP2), FIP3 (si-FIP3), α5 integrin (si-α5) or a non-targeting control (si-Nt). Transfected cells were sparsely seeded onto glass-bottomed wells coated with 33% Matrigel. Cells were serum-starved for 24 h and then treated with HGF (10 ng ml−1) to initiate cell migration. Cell movement was recorded using time-lapse video microscopy with frames being collected every 5 min. Representative movies illustrate collisions between control (si-Nt; movie 1) and RCP knockdown (si-RCP; movie 2) cells and frames from these are displayed in a. The frames have been aligned so that the start of the cell collision is at t=0 min. The green arrows indicate the direction from which the cells approached one another, and the red arrows indicate the direction of migration after the collision. The time that cells spent touching each other during collision was determined for all collisions in which only two cells were involved and both cells were migrating towards each other pre-collision (b; left panel and c). The speed of cell migration between collisions was also calculated from these movies (b; right panel). Bar, 10 μm. Data are represented as box and whiskers plots (whiskers: 10–90 percentile,+represents the mean). ***P<0.001, *P<0.01; one-way ANOVA (Kruskal-Wallis test, Dunn's Multiple Comparison Test). Data are from three independent experiments with >50 collisions being tracked per condition. (d–f) H1299 cells were transfected with siRNAs targeting RCP (SMARTPool (si-RCP) or an individual oligo (si-RCP#1), individual oligos targeting EphA2 (EphA2#1 and EphA2#2) or a non-targeting control (si-Nt). Transfected cells were seeded onto plastic surfaces and allowed to grow overnight to form colonies of approx. 4 cells per colony. Cells were then treated with HGF (10 ng ml−1) and cell scattering was recorded using time-lapse video microscopy with frames being collected every 5 min over a 6 h period. Representative trackplots of movies illustrate the scattering reaction of control (si-Nt), EphA2 knockdown (si-EphA2) and RCP knockdown (si-RCP) cells (d). Cell scattering was quantified using ImageJ manual tracking and chemotaxis plugin, and is expressed as the accumulated distance travelled over 6 h (e,f). Data are represented as box and whiskers plots (whiskers: 10–90 percentile,+represents the mean). ***P<0.001; one-way ANOVA (Kruskal-Wallis test, Dunn’s Multiple Comparison Test). Data are from three independent experiments with >50 cells being tracked per condition. Bar in d, 50 μm.