Figure 4: DUB3 regulates EMT through SNAIL1.

(a) T47D and MCF-7 cells were transfected with indicated plasmids and western blot was performed with indicated antibodies. (b) T47D cells were transfected with indicated plasmids and cell morphological changes associated with EMT are shown in the phase contrast images. Expression of E-cadherin and vimentin was analysed by immunofluorescence. Nuclei were visualized with 4,6-diamidino-2-phenylindole staining (blue). Scale bars, 25 μm. (c–e) MDA-MB-231 cells were stably transfected with control and DUB3 shRNAs (c). (d) The migratory ability of cells was analysed by wound-healing assay and results are quantified in e. The results represent the means±s.d. of three independent experiments. ^##P<0.01. (f) The invasiveness of cells was analysed with a chamber invasion assay. The results represent the means (±s.d.) of three independent experiments. ^##P<0.01. (g–j) One million cells from c were injected into the lateral tail vein of immunodeficient mice (n=8). After 6 weeks, the development of lung metastases was recorded using (g) bioluminescence imaging and (h) quantified. After 12 weeks, mice were killed and lung metastatic nodules were counted and quantified (i,j). Data are expressed as the means±s.d. Statistical analyses were performed with the analysis of varinace. ^##P<0.01.