Ectopic expression of BST-2 inhibits MMTV replication in tissue culture cells. (A-B) Varying concentrations of BST-2 were transiently transfected into TRH3 cells. Twenty four hours later, (A) a portion of cells were processed for FACS analysis and evaluated for BST-2 surface expression and (B) the remaining portion was infected with MMTV and examined for level of infection by qPCR 24 hours after infection. Data are normalized to GAPDH and presented as fold change in viral DNA relative to viral DNA of vector transfected cells. (C-E) Constructs of BST-2 and puromycin were stably co-expressed in TRH3 cells. Twenty four hours later, fresh medium containing puromycin drug was added to cells to facilitate the selection of transfectants. After about 10 days in selection, individual colonies of transfected cells were picked and amplified. A portion of cells were examined for (C) BST-2 surface expression by FACS. Another portion of cells were infected with MMTV-gfp. Twenty four and ninety six hours after infection, cells were analyzed for infection as determined by (D) level of gfp expression or (E) intensity of gfp expression using FACS. Error bars are standard deviation, p is significance level. All experiments were repeated at least three different times with similar results.