VAPB-dependent cell growth is mediated through AKT activity.
(A) MCF-10A cells expressing VAPB or carrying control vector were serum starved and stimulated with 20 ng/mL EGF at the indicated time points. Phospho-AKT levels in MCF10A-HER2 cells were measured by western blot analysis. (B) VAPB was knocked down in MMTV-Neu cells (KD#1) and re-expressed via retroviral transduction (KD#1_VAPB). Phospho-AKT levels were measured by western blot analysis Representative blots from 3 independent experiments are shown. (C–E) Pharmacologic AKT inhibition significantly impaired VAPB-mediated spheroid growth in MCF10A-Her2 cells (C) and MMTV-Neu VAPB knockdown cells rescued with VAPB reexpression (E) (p<0.05 ANOVA). Inhibition of AKT activity was confirmed by western blot analysis for phospho-AKT in MCF10A-HER2 cells (D) and MMTV-Neu knockdown cells (F).