UEV 1A overexpression promotes breast cancer cell invasion in vitro and metastasis in a xenograft model. (A) pcDNA4.0/TO/HA(+) vector (CK) expressing UEV1A, UEV1C or MMS2 was stably transfected into MDA-MB-231-TR cells, with or without doxycycline (Dox) treatment. The level of ectopic gene expression was monitored by western blot against an anti-HA antibody. (B) Representative images of wound-healing assays with Dox treatment. (C) Statistical analysis of cell migration of wound-healing assay with and without Dox treatment. The migration distance of cells was measured in five different wells in each group under a light-microscope. (D) Representative images of cell invasion assay with Matrigel-coated transwells. (E) Statistical analysis of the cell invasion assay data. Cells that invaded the lower surface of the filter were counted in five random fields under a light-microscope at 200× magnification. (F-I) In vivo tumorigenesis and metastasis assays using a xenograft mouse model. (F) Lymph node sections after sacrifice were stained with H&E. The lymph node metastasis sites are shown by red arrows. (G) Quantitative analysis of tumor growth. Tumor growth was measured every week after injection (Day 0) and expressed as mean ± SD (n = 10). (H) The in vivo metastasis assay in xenograft mice. Upper panel, the lung metastasis nodules formed are shown by red arrows. Lower panel, the lung sections were stained with H&E and the lung metastasis under a light-microscope at 100× magnification is indicated by a red arrow. (I) Quantitative analysis of the in vivo lung metastasis as measured by the number of metastasis foci per lung for all four sections (n = 10 mice for each treatment).