Figure 3
Polycystin-1 regulates cell adhesion to the substrate. (A) Images of the colorimetric adhesion assay of MDCKZeo (clones F2, F6) and MDCKPKD1Zeo (clones 36, 68) cells upon wash-out. As control, images of unwashed cells are shown. (B) Experiments as in A are quantified by measuring absorbance (OD 570 nm) of colored cells revealing that MDCKPKD1Zeo cells adhere more to fibronectin. The graph is representative of four independent experiments; averages and SD are shown. Statistical analysis: ANOVA; *p < 0.05, **p < 0.01, relative to both control clones (F2, F6). (C-E) Time course of a colorimetric adhesion assay with: MDCKZeo (clone F2) and MDCKPKD1Zeo (clone 68) cells (C), Pkd1 +/+ and Pkd1 −/− fibroblasts (D), mIMCDshCtrl (clones M3, M4) and mIMCDshPkd1 (clones C12, C16) cells (E); cells plated on fibronectin were washed at the indicated time points and values normalized for the 6 h unwashed values. Graphs are representative of three independent experiments, performed in triplicate; averages and SD are shown. Statistical analysis: ANOVA; NS non-statistically significant (p > 0.05), *p < 0.05, **p < 0.01, ****p < 0.0001, referred to the control (F2, Pkd1 +/+, mIMCDshCtrl) relative bars. (F) Quantification of the colorimetric adhesion assay in MDCKZeo (clone F2) and MDCKPKD1Zeo (clone 68) cells, or in Pkd1 +/+ and Pkd1 −/− fibroblasts, in presence of cytochalasin (cyt, 5 μM) and latrunculin (lat, 5 μM). revealing an essential role of actin in adhesion. Graph is representative of three independent experiments. Averages and SD are shown. Statistical analysis: ANOVA; NS non-statistically significant (p > 0.05), *p < 0.05, ****p < 0.0001, referred to the relative control (ct). (G) Staining of MDCKZeo (clone F2) and MDCKPKD1Zeo (clone 36) cells for actin (phalloidin-TRITC, red), paxillin (green) and nucleus (DAPI, blue) after 1 h on fibronectin. Zoom-in of the boxed areas are shown. Bar: 25 μm.