Figure 6
Polycystin-1 induces Focal Adhesion Kinase (FAK) activation, important for cell adhesion, migration and front-rear polarity. (A) Western blot analysis in MDCK cells (left panel) and fibroblasts (right panel), after overnight plating at 50% confluence reveals higher phosphorylation, i.e. activation, of FAK in MDCKPKD1Zeo cells compared to MDCKZeo, and lower in Pkd1 −/− compared to Pkd1 +/+ fibroblasts. (B-C) Western blot analysis as in A of cells collected at different time points after wounding (B) or after plating (C). (D) Colorimetric adhesion assay performed in the presence of the FAK inhibitor PF-228 (PF, 10 μM) revealed a significant decrease in adhesion of both Pkd1 +/+ and MDCKPKD1Zeo cells. Graphs are representative of three independent experiments. Averages and SD are shown. Statistical analysis: ANOVA; NS (non-significant) p > 0.05, **p < 0.01, ***p < 0.001. (E) Boyden chamber assays performed in presence of PF-228 (PF, 10 μM) results in a significant decrease in migration of Pkd1 +/+ and MDCKPKD1Zeo cells. Averages and SD are shown. Statistical analysis: ANOVA; NS (non-significant) p > 0.05, *p < 0.05. (F) Golgi orientation in wound-healing assays in the presence of PF-228 (PF, 10 μM) show a significant decrease in front-rear polarity of Pkd1 +/+ fibroblasts and MDCKPKD1Zeo cells. Averages and SD are shown. Statistical analysis: ANOVA; NS (non-significant) p > 0.05, **p < 0.01, ****p < 0.0001. (G) PF-228 (PF, 10 μM) significantly decreases focal adhesion disassembly in MDCKPKD1Zeo cells evaluated by nocodazole washout experiments. The graph is representative of three independent experiments. Averages and SD are shown. Statistical analysis: ANOVA; NS (non-significant) p > 0.05, ***p < 0.001, referred to the relative control (ct) bar. (H) Western blot analysis reveals that PF-228 (PF, 10 μM) inhibits phosphorylation of the PI3kinase target Akt, while Wortmannin (WTM, 2.5 μM) does not affect FAK phosphorylation.