Deregulated c-Myc enhances the c-Fos apoptotic pathway. (A) Cell viability. Cells were seeded at 0.1 × 10⁶ cells/mL in the absence or presence of estrogen. At each time point cell viability was determined by trypan blue dye exclusion as described in “Materials and methods.” Data shown are representative of at least 3 experiments that were conducted using MlFosER-myc clones 4 and 5. (B) DNA fragmentation. At least 10⁷ cells were harvested at indicated times after treatment with estrogen, and high-molecular-weight DNA was extracted and resolved on a 2% agarose gel using 10 μg/lane, as described in “Materials and methods.” Similar data were obtained using MlFosER-myc clones 4 and 5; results are shown for clone 4. (C) Analysis of procaspase-9 cleavage. Protein lysates were collected from cells at designated time points after treatment with estrogen and resolved on a 10% SDS-PAGE gel using 50 μg/well. Gels were transferred to PVDF membranes (Millipore) and probed with primary antibody recognizing both the pro and the cleaved forms of caspase-9 (4 μg/mL; Stressgen). Signal was detected using an HRP-linked secondary antibody (Santa Cruz Biotechnology) and the Pierce detection system. (D) Analysis of cytochrome c release. Cytospin smears of untreated and βE2-treated MlFosER cells were analyzed for the redistribution of cytochrome c using immunohistochemistry with an anti-cytochrome c FITC-conjugated antibody (Stratagene) at 1 μg/mL (photomicrographs, original magnification × 400). In addition, at the indicated time points, 2 × 10⁷ cells were collected and lysed. After pelleting of unlysed cells and nuclei, the mitochondrial fraction (M) and cytosolic supernatant fraction (S) were separated by centrifugation at 13 000g. For Western blot analysis, 10 μg mitochondrial and 20 μg supernatant fraction were resolved on a 15% SDS-PAGE gel, and an anti-cytochrome c antibody (murine; Pharmingen) was used to detect cytochrome c.