Fig. 4. mitoERβ inhibits the tumorigenesis of breast cancer cells in vitro and in vivo. A. TNBC cell lines were transfected with pHA-mito-ERβ or Mock, and the cells were subjected to colony-forming assays. The numbers of colonies generated per 10,000 cells were counted 3 weeks later. B. ERβ-overexpressing and control cells were subjected to sphere-formation assays in ultra-low-attachment 96-well plates after 72 hrs. The numbers of spheroids generated per 10,000 cells were counted 2 weeks later. C-E. To determine the effects of mitoERβ on the kinetics of tumor growth, five mice each were implanted with ERβ-overexpressing and control cells. Images of tumors taken from the mice in each group are presented, with the data representing the mean ± standard error of tumor volumes recorded at 3 day intervals (C). Tumor samples from each mouse were subjected to immunoblotting with anti-ERβ antibody (D). Kaplan-Meier analysis of overall survival over time of the animals in the two groups, with between-group differences determined by the log rank test (E).