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. 2007 Sep 17;27(22):7906–7917. doi: 10.1128/MCB.01369-07

FIG. 8.

FIG. 8.

PI3K-C2β and AKT are epistatic to ITSN. (A) Wild-type N1E-115 cells were differentiated in the presence of PI3K inhibitor (LY294002) or control vehicle (DMSO), and cell survival was quantified using an ATP assay. (B) N1E-115 cells were stably transfected with either empty vector or a kinase-dead AKT construct (AKT-DN). Cells were grown in the presence of serum (top panels) or differentiated by removal of serum (bottom panels). (C) Parallel cultures from the samples shown in panel B were incubated with CellTiterGlo reagent to quantify cell survival. Expression of kinase-dead AKT (AKT DN) resulted in increased cell death. (D) ITSN-silenced cells (M1635) were stably transfected with either CFP or CFP-PI3K-C2β. Cells were then grown in the presence of serum or differentiated by removal of serum. (E) ITSN-silenced cells (M1635) were stably transfected with either empty vector or HA-tagged wild-type (AKT-WT), activated (Myr-AKT), or kinase-dead (AKT-DN) AKT. Cells were then grown in the presence of serum (top panels) or differentiated by removal of serum (bottom panels). (F) Survival of cells shown in panel E was determined using CellTiterGlo reagent as described for panel C. (G) Expression of various HA-tagged AKT proteins was determined by Western blot analysis of cell lysates with anti-HA antibodies (top panel). These same lysates were also analyzed for expression of ITSN. pSR, N1E-115 cells stably transfected with empty pSuperRetro vector; M1635, N1E-115 cells stably transfected with the pSR-M1635 shRNA construct that silences ITSN expression. The M1635 cells were also stably transfected with AKT expression constructs (wild type [WT], kinase dead [DN], or activated [Myr]) or empty vector (vector). α, anti.