Fig. 6.

Reduction of the abundance of LAT2 by ODPC or LAT2 knockdown by shRNA (shRNA LAT2) is associated with lack of activation of the AKT pathway. A, ODPC (25 μm) reduced AKT and phospho-AKT levels after 3 h of treatment; however, PTEN was reduced only at 24 h, when apoptosis was fully installed. ODPC treatment induced a state of hypophosphorylation at the C terminus of PTEN, which has been reported to be required in order to initiate phosphatase activity (41). B, ODPC inhibited AKT activation by myeloid growth factors (MGFs) in a manner similar to Wortmannin, a specific PI3K inhibitor. ODPC inhibited the up-regulation of LAT2 induced by MGFs under the same conditions and inhibited ribosomal S6P phosphorylation induced by MGFs. NB4 cells were maintained serum-free (SF) overnight, treated with PBS as control with 25 μm ODPC or 1 μm Wortmannin for 15 min, stimulated with an MGF mixture (10 ng/ml IL-3, GM-CSF, l-FLT-3, and SCF), and harvested at the indicated times. C, LAT2 stable knockdown was obtained from NB4 cells via lentiviral transduction of shRNA that targets LAT2. D, AKT activation was suppressed in LAT2 stable knockdown NB4 cells. Both cell lines were maintained SF overnight and subsequently stimulated with an MGF mixture (same as described above) and harvested at the indicated times.