MCF7 and SUM149 cells were cultured per American Type Culture Collection (ATCC) recommendations. Primary murine mammary tumor cells were cultured in phenol-free DMEM/F12 (Gibco), with 10% charcoal-stripped FBS (Gibco), 10 μg/ml insulin, and 10 ng/ml EGF. For treatment of estrogen, AZD5363, and 4OHT, tumor cells were cultured in 10% charcoal-stripped FBS in the presence of E2, AZD5363 (ApexBio Technology), 4OHT (Sigma), or dimethyl sulfoxide (DMSO) for the indicated times and then collected for further analysis. To determine cell viability, 50,000 cells were plated in 24-well plates and treated with DMSO or drugs at the indicated concentrations for 5 days. Viable cell numbers were determined on day 1, day 3, and day 5 by an automatic cell counter (Bio-rad) with trypan blue exclusion. For western blot, tissue and cell lysates were prepared as previously reported [19 (link), 23 (link), 37 (link)]. Primary antibodies used are as follows: HSP90 (Santa Cruz), Gapdh (Ambion), ERα (Santa cruz), Brca1, p-Akt (Ser473), p-4E-bp1 (Thr37/46), p-mTor (Ser2248), p-Gsk3β (Ser9), E-cad, Vim, Snail, Slug, p-Fra1 (Ser 265), p-Rb (ser780) (Cell Signaling) and Fn (Abcam).
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