Catalytic AKT inhibitor suppresses LTED growth and prevents emergence of hormone-independent breast cancer cells. A) LTED cells were treated with 10% DCC-FBS ± 0 to 15 µM AZD5363 for 24 hours. Protein lysates were analyzed by immunoblot using the indicated antibodies. B) LTED cells were treated with 10% DCC-FBS ± 0.4 or 2 µM AZD5363. Media and drugs were replenished every three days. Cells were counted after five to ten days. Data are presented as percent of control; each bar, mean ± SEM (n = 3; *P <0.0001 versus Con, one-way ANOVA). C) Parental cells in 10% DCC-FBS were treated with ± 2 µM AZD5363 or 1 µM selumetinib. Media and drugs were replenished every three days. When control monolayers reached 60% to 80% confluency (after 15 (MCF-7), 36 (ZR75-1 and MDA-361) or 38 (HCC-1428) days, respectively), cells were fixed and stained with crystal violet. Representative images and quantification of integrated intensity (% control) are shown (*P <0.05 versus control, t-test). ANOVA, analysis of variance; DCC-FBS, dextran/charcoal-treated fetal bovine serum; LTED, long-term estrogen deprivation; SEM, standard error of the mean.