Inhibition of phosphatidylinositol-3-kinase pathway signaling in estrogen-independent long-term estrogen-deprived cells. (a) Western blot showing estrogen receptor (ER) expression and levels of p-Akt, p-S6 and p-ERK1/2 in estrogen-deprived parental MCF7 and T47D cells and in estrogen-deprived long-term estrogen deprivation (LTED) and estrogen-retreated long-term estrogen-deprived (LTED-R) sublines of each parental cell line. Equal amounts of total protein (25 μg) were immunoblotted for the indicated proteins. Total Akt, S6, ERK1/2 and actin are shown as loading controls. Representative results obtained from at least two different protein isolates per cell line are shown. (b) Western blot showing effects of BGT226, BKM120 and RAD001 on p-Akt and p-S6 in MCF7 LTED and T47D LTED lines. Representative results obtained in at least two experiments per cell line per drug treatment are shown. Estradiol (E2) stimulates the growth of MCF7 and T47D cells, but not (c) MCF7 LTED cells and (d) T47D LTED cells. Cells were treated with or without the indicated concentrations of E2 for 7 days followed by growth measurement. Cells were treated with fulvestrant (300 nmol/l) to directly inhibit ER function. Values are normalized to untreated cells. Shown are results from representative experiments performed in triplicate. Fulvestrant inhibited the growth of MCF7 LTED cells (*P < 0.05). DMSO, dimethylsulfoxide; LY, LY294002.