Activation of FosER in MlFosER cells results in growth retardation accompanied by suppression of c-Myc and apoptotic cell death that is abrogated by IL-6. (A) Untreated and estrogen-treated cells were collected 24 hours after initiation of treatment and subjected to FACS analysis as described in “Materials and methods.” Data presented are an average of at least 3 experiments, with each yielding similar results. (B). Cells were collected at the indicated time points after treatment with estrogen, and total RNA was extracted as described in “Materials and methods.” RNA (10 μg/lane) was resolved on a 1% agarose formaldehyde gel and transferred to a Duralon-UV membrane (Stratagene). RNA blots were hybridized to ³²P-labeled c-Myc cDNA probe, washed, and subjected to autoradiography for 24 to 48 hours at -80°C as described in “Materials and methods.” (C) Cells were seeded at 0.1 × 10⁶ cells/mL with estrogen, IL-6, or with both estrogen and IL-6, and viability was assessed by trypan blue dye exclusion as described in “Materials and methods.” Each time point represents the average of at least 3 experiments, with a standard deviation of up to 10% (ie, 40% + 4%). (D) For DNA fragmentation analysis, high-molecular weight genomic DNA was extracted from 1 × 10⁷ cells at indicated time points and resolved on a 2% agarose gel using 10 μg/lane as described in “Materials and methods.” (E) Untreated and estrogen-treated cells were collected at indicated time points and lysed with RIPA buffer. Total protein extracts were resolved on a 7.5% SDS-PAGE gel using 50 μg/well, transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore; Billerica, Spain), and probed with anti-PARP antibody (0.5 μg/mL) in 5% milk. Signals were developed by using ECL Western blotting as described in “Materials and methods.”