Figure 6: NEDD4 is involved in the cell-density-dependent activation of the Hippo pathway.

(a) NEDD4 modulates the density-dependent subcellular localization of YAP. GFP-YAP was transfected in NIH3T3 control cells or NIH3T3-NEDD4 KD cells. At 24 h post transfection, cells were cultured as either sparse subconfluent or dense confluent monolayers. Cells were subjected to immunofluorescence staining and the subcellular localization of YAP and NEDD4 was visualized under a LSM710 multi-photon confocal laser scanning microscope (Zeiss). The scale bar represents 10 μm. (b) Cell density affects the protein levels of the Hippo pathway components as well as NEDD4. Equal numbers of NIH3T3 cells (1.3 × 10⁵) were seeded in a different size of culture dishes to achieve the different cell density (L, low cell density to 100 mm dish; M, medium cell density to 60 mm dish; H, high cell density to 35 mm dish). After 24 h incubation, cells were subjected to immunoblotting. Relative protein levels quantitated by ImageJ and normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) are shown (n=3). (c) WW45 and LATS2 protein levels remain unchanged regardless of cell density in NEDD4 KD stable cells. Control or NEDD4 KD NIH3T3 cells were cultured sparsely or to maximum confluence for 24 h and then subjected to immunoblotting. Relative protein levels quantitated by ImageJ and normalized to β-actin are shown (n=3). (d) Expression of YAP-controlled genes is inhibited in NEDD4 KD cells. Total RNA isolated from either control or NEDD4 KD stable NIH3T3 cells was used for quantitative real-time RT–PCR analyses. Normalization was carried out with respect to the mean values of β-actin and hypoxanthine phosphoribosyltransferse (HPRT). Data are plotted as the mean±s.e.m. of five independent experiments and analysed by one-way analysis of variance (ANOVA) with Bonferroni’s multiple comparison test (***P<0.0001). (e,f) Growth suppression of NEDD4 KD cells was partially recovered by simultaneous knockdown with either siWW45 or siLATS2 in NEDD4 KD cells. (e) Control or NEDD4 KD stable cells were transfected with indicated siRNAs and replated for the subsequent proliferation assay. Data are plotted as the mean±s.e.m. of four independent experiments and analysed by one-way ANOVA with Bonferroni’s multiple comparison test (***P<0.0001). (f) Control or NEDD4 KD stable cells were transfected with indicated siRNAs. At 24 h post transfection, 150 cells were seeded into six-well plates and formed colonies were scored after 10 days. Data are plotted as the mean±s.e.m. of three independent experiments and representative plates are shown. DAPI, 4',6-diamidino-2-phenylindole.