Depletion of ubiquitin conjugating enzyme variant (Uev)1 reduces cell invasion in vitro and metastasis in a xenograft mouse model. (A) MDA-MB-231 cells were transfected with shRNA lentiviral particles against UEV1 (shUEV1) or non-specific target (shCK). shUEV1-1 and shUEV1-2 represent two independent stable shUEV1 lines. UEV1A transcript levels in shCK and shUEV1 lines were determined by qRT-PCR. (B) Quantitative analysis of cell migration by wound-healing assay. The migration distance of cells was measured in five different wells in each treatment group under a light-microscope. (C) Quantitative analysis of cell invasion in Matrigel-coated transwells. Cells invading the lower surface of the filter were counted in five random fields under a light-microscope at 200× magnification. (D, E) The in vivo tumorigenesis assay using a xenograft mouse model. 1 × 106 MDA-MB-231 cells depleted with shUEV1 or shCK were injected into the lateral flanks of 4- to 5-week-old BALB/c female nude mice. (D) Tumor growth was measured every week after injection (Day 0) and expressed as mean ± SD (n = 10). (E) Upper panel, lungs from mice injected via tail veins with MDA-MB-231 cells treated with shUEV1s or control. Red arrows point to lung metastasis foci. Middle panel, quantitative analysis of the number of metastasis foci per lung. The nodules per lung for all four sections were counted under a light-microscope (×100) (n = 10 mice for each treatment). Lower panel, sample lung sections stained with H&E. Red arrows point to lung metastasis foci. All samples were taken after sacrifice.