Figure 10: PtdIns5P is required for invasion of dense Matrigel and invadosome formation.
From: Phosphatidylinositol 5-phosphate regulates invasion through binding and activation of Tiam1
(a) Invasiveness of transduced RPE-1 cells into 3D-Matrigel toward a FBS gradient or FGF-1 was examined using transwell chambers. The number of cells penetrating the Matrigel was expressed as a fold increase compared with the experiment without serum gradient. Data are shown as mean±s.e.m. (n=3; *P<0.05 using non-parametric one way ANOVA (Kruskal–Wallis) with Dunn’s post test) (b) Same experiment as in (a) except that RPE-1 were treated overnight with NSC23766 (100 μM). Data are shown as mean±s.e.m. (n=3; *P<0.05 using non-parametric one way ANOVA (Kruskal–Wallis) with Dunn’s post test). (c) Karpas 299 or Cost cells were treated overnight with NSC23766 (40 μM) before CMTMR staining. Invasiveness towards an FBS and 1 μg ml−1 SDF1α gradient was examined. The number of cells penetrating the Matrigel to depths of 20 μm and greater was expressed as a percentage of the total cell number within the gel. Data are shown as mean±s.e.m. (n=3; *P<0.05 using non-parametric one way ANOVA (Kruskal–Wallis) with Dunn’s post test). (d) IpgD or Inp54p expression was induced in MEF Tet-Off cells before serum starvation. Then, invasiveness into 3D-Matrigel ±NSC23766 (100 μM) towards an FBS gradient was examined. Data are shown as mean±s.e.m. (n=3; *P<0.05 using non-parametric one way ANOVA (Kruskal–Wallis) with Dunn’s post test). (e) Serum-starved control, IpgD or Inp54p-expressing MEFs were seeded into transwell chambers. Migration toward a gradient of FBS was allowed for 6 h ±NSC23766 100 μM and the percentages of migrated cells were determined. Data are shown as mean±s.e.m. (n=3 experiments; *P<0.05 using non-parametric one-way ANOVA (Kruskal–Wallis) with Dunn’s post test). (f) MEF-IpgD were cultured on FITC-gelatin-coated coverslips for 24 h. Degradation areas were visualized as dark holes. z projections show that actin cores are present within the degraded gelatin. The percentage of cells that presented at least one area of gelatin degradation was determined. Data are shown as mean±s.e.m. (n=3).
