Target validation of CIP2A-Akt-4EBP1.

<p>A, ectopic expression of CIP2A-myc abolishes effects of bortezomib on autophagy in Huh-7 and Hep3B cells. Cells were transfected with CIP2A-myc and were selected for 8 weeks by G-418. Analysis of autophagy was performed by western blot (WB) after cells were sequentially exposed to DMSO or bortezomib (200 nM or 400 nM or 800 nM) for 24 hours. B, <i>left,</i> co-treatment with calyculin A, a PP2A inhibitor, reverses the effect of bortezomib on CIP2A, P-Akt, P-4EBP1 and autophagy. Cells were treated with calyculin A (1 mM) or bortezomib (800 nM) for 24 hours. <i>Middle,</i> ectopic expression of Akt1 reduced the effects of bortezomib on autophagy in Huh-7 cells. <i>Right,</i> ectopic expression of 4EBP1 reduced the effects of bortezomib on autophagy in Huh-7 cells. C, <i>left,</i> co-treatment with rapamycin, a mTOR inhibitor, enhanced bortezomib-induced autophagy in Huh-7 cells. Cells were treated with rapamycin (100 nM) and/or bortezomib (400 nM) for 24 hours. <i>Right,</i> Co-treatment with autophagy inhibitor 3-Methyladenine (3-MA) reduced bortezomib-induced autophagy. Huh7 cells were treated with bortezomib (800 nM) and/or 3-MA (1 mM) for 16 hours.