Fig. 7.
Impact of AKT and MEK inhibition on PI3K-AKT and MAPK signaling pathways and proliferation in non-malignant breast epithelial cells expressing mutant HRASQ61R. a Representative western blot analysis of p-ERK1/2 (T202/Y204), p-p90 RSK (S380), p-AKT (S473), p-AKT (T308), p-PRAS40 (T246), p-FOXO1/3a/4, p-GSK3β (S9), p-mTOR (S2448), p-p70 S6K (T389), and p-S6 (S240/244) protein in MCF-10AP and MCF-10AH1047R cells stably expressing empty vector (EV) or mutant HRASQ61R treated with 2 µM AKT inhibitor (AKTi, MK2206) at different time points. β-actin was used as a protein loading control. Experiments were repeated at least twice with similar results. b Cell proliferation assay of MCF-10AP and MCF-10AH1047R cells stably expressing EV or mutant HRASQ61R. ****P < 0.0001; two-tailed unpaired t-test. c Inhibition effects of cells treated with DMSO (black), 1 µM AKTi (blue), 10 nM MEK inhibitor (MEKi, GSK212, green), and combination of 1 µM AKTi and 10 nM MEKi (red) for 3, 5, and 7 days. In b and c, cells were cultured in growth factor- and serum-free media. Data are representative of three independent experiments. Error bars, s.d. of mean (n = 3). d Representative micrographs of MCF-10AP and MCF-10AH1047R cells stably expressing EV or mutant HRASQ61R cultured after 3 days and treated with DMSO, 1 µM AKTi, 10 nM MEKi, or combination of 1 µM AKT and 10 nM MEK inhibitors for 6 and 9 days are shown (scale bars, 400 µm). DMSO or inhibitors were added after seeding the cells in three-dimensional basement membrane for 3 days; fresh media with DMSO or inhibitors was replenished every 3 days. Triplicate experiments were repeated at least twice with similar results