Estrogen treatment and growth curve analysis
G418-resistant colonies from BRCA1 antisense infected BG-1 cells and control vector LXSN-infected BG-1 cells were pooled, pretreated for 5 days in phenol red-free, DMEM/F-12 medium (Gibco/Life Technologies) supplemented with 10% charcoal/dextran treated serum (Hyclone, Logan, UT, USA), then plated at 2.5 × 106 cells per 100mm dish in triplicate for growth curve analysis in the absence or presence of estrogen (10-8mol/l; 17β-Estradiol; 1,3,5 (10) - Estratriene 3, 17β-diol; Sigma, St Louis, MO, USA). Extended growth curve analysis was plated at 2.5 × 106 in 100mm dishes for an extended treatment of 20 days without estrogen. Clones were isolated from the BRCA1 antisense and LXSN BG-1 pooled populations and grown in phenol red-free, DMEM/F-12 medium (Gibco/Life Technologies) supplemented with charcoal/dextran treated serum (Hyclone) for 5 days before plating for growth curve analysis. Cells were then plated in triplicate at 1 × 106 cells per 60mm dish in either the absence or presence of estrogen (10-8 mol/l) and grown for 8-10 days. Cell number was calculated on indicated days using a Coulter counter. The number of dead cells for the extended growth curve experiment was calculated by counting trypan blue incorporated cells using a hemocytometer. Statistical analyses of P values were calculated based on the fold differences between growth of the clones by computing a mean ratio and the corresponding standard deviation [33].