Figure 2. Disruption of RAS interaction with PI3-Kinase disturbs cell polarity and invasion.

(a) Wounded Pik3ca^WT and Pik3ca^RBD MEFs monolayers were allowed to migrate for 18 h in the presence or absence EGF (20 ng ml⁻¹) or FBS (10%). Migration was analysed by time-lapse video microscopy. For each condition 90 cells were tracked and persistence in the directionality of migration was analysed using Mathematica software. Analysis of variance (ANOVA) statistical analysis was performed with starved cells used as reference for each condition (NS, not significant; **P<0.05; ***P<0.005). (b) Invasion of Pik3ca^WT and Pik3ca^RBD cells in a collagen I matrix in the presence or absence of EGF (0.5 μg ml⁻¹). Stacks are acquired from the bottom of the well over 150 μm upward. Invasion through the collagen layer was monitored in a confocal microscope and analysed using Mathematica software. ANOVA statistical analysis was performed (**P<0.05; ***P<0.005). (c) Invasion of Pik3ca^WT, Pik3ca^RBD and Pik3ca^RBD WT p110α MEFs in transwells containing a layer of matrigel (growth factor reduced matrigel). Invasion was measured in either 0.2% FBS (starved), EGF (50 ng ml⁻¹) or FBS (10%). Invasive cells (on the lower part of the transwell, attached to the membrane) were stained with crystal violet and then lysed using acetic acid. Assays were carried out in triplicate, with error bars indicating s.d. The results of two different experiments are shown. Error bars indicate s.d. (Significance using Student's t-test. NS, not significant; **P<0.05; ***P<0.005).