Agent concentrations were as follows: fulvestrant, 100 nM; MEK inhibitor U0126, 1 μM; PI3K inhibitor LY294002, 1 μM; lapatinib, 1 μM. (A) Data are shown as means ± SD of three independent experiments, relative to cells treated with vehicle. *P<0.01 in kinase inhibitor monotherapy and in combination with fulvestrant in EvR. Protein levels in Type 1 EDR-V1 and EvR-v1 cells treated with fulvestrant (100 nM) or U0126 (1 μM). (B) Protein was extracted 24 h after each agent was added. p-MAPK was not affected by either treatment, but fulvestrant inhibited total ERα expression in each cell type. p-Elk-1, downstream of ER, was unaffected by treatment. Mechanism of acquired resistance to everolimus in Type 1 cells. (C) After resistance to everolimus was acquired, cell proliferation depended on the MEK/MAPK cascade, and not the PI3K/Akt/mTOR cascade.