Fig. 5.

The induction of cell migration and invasion by DEPTOR depletion is abrogated by the AKT inhibitor MK-2206. a DEPTOR knockout causes the accumulation of Snail. Cells transfected with the indicated sgRNA were subjected to western blotting using the indicated antibodies. b, c DEPTOR knockout has minor effects on Snail mRNA levels, but extends Snail protein half-life. Cells were harvested for RT-qPCR analysis (b), or treated with CHX for various time periods, followed by western blotting (c). Densitometry quantification was performed with Image J, and the decay curves are shown (right, c). d DEPTOR knockout increases nuclear translocation of β-catenin. Cells transfected with the indicated sgRNA were subjected to nuclear fractionation, followed by western blotting using the indicated antibodies. PARP and Pro-caspase 3 served as markers of the nuclear and cytoplasmic fractions, respectively. e, f MK-2206 treatment abrogates the induction of cell migration and invasion by DEPTOR depletion. Cells were treated with MK-2206 or left untreated, and then subjected to transwell migration (e) and invasion (f) assays. Representative images of migratory and invasive cells are shown (left, e and f). The number of migratory and invasive cells was counted in five random fields per chamber insert (right, e and f). The mean ± SEM from three independent experiments are shown; n = 3; *p < 0.05, **p < 0.01; ***p < 0.001. g The induction of Snail upon DEPTOR knockout is reversed by MK-2206 treatment. Cells were treated with MK-2206 for 12 h or left untreated, followed by western blotting. h β-catenin nuclear translocation induced by DEPTOR knockout is partially inhibited by MK-2206 treatment. Cells were treated with MK-2206 or left untreated and then subjected to nuclear fractionation, followed by western blotting. The band density was quantified and expressed as the relative gray value (compared with the control), by arbitrarily setting the control value as 1