Ectopic Bc1-2 protects MlFosER cells from Fos-mediated apoptosis. (A) MlFosER/Bcl-2 cell lines were established by infecting MlFosER cells with MSCV-Bcl-2/puro retroviral vectors followed by selection of cells in G418 and puromycin as described in “Materials and methods.” At designated time points after estrogen addition MlFosER/Bcl-2 cells (clone 2) were collected, lysed in RIPA buffer, resolved on a 12.5% SDS-PAGE gel, and analyzed for Bc1-2 expression by Western blotting. After blocking in 5% milk, blots were incubated with 1 μg/mL anti-Bcl-2 primary antibody (Santa Cruz Biotechnology; 1:5000), and the signal was detected using the ECL system. (B). Indicated cells were seeded at 0.1 × 10⁶ cells/mL in the presence and absence of estrogen, and viability was assessed by trypan blue dye exclusion. Percent viability at each time point is an average of at least 3 experiments, with a standard deviation of up to 12% (ie, 30% ± 3.6%). (C) For analysis of DNA ladders, high-molecular weight DNA was extracted from 1 × 10⁷ cells at indicated time points after treatment with estrogen (2 mM), and 10 μg/lane was resolved on a 2% agarose gel as described in “Materials and methods.”