Fig. (4). Expression of PCDH24 inhibits PI3K activity in HCT116-PCDH24-HaloTag cells and the over-expression of galectin-1 and -3 prevents this inhibition (a) PI3K activity was assessed using quenched fluorescence signals. Whole cell lysate was extracted and the fluorescence signal was measured. As a control, the PI3K inhibitor LY294002 was used. HCT116-PCDH24-HaloTag cells were exposed to LY294002 (100 µM) for 48 h before the assay. HCT116-PCDH24-HaloTag cells were cultured with or without DOX. Galectin1 and galectin3 indicate cells that were transiently transfected with the HaloTag-fused galectin-1 or galectin-3 expression clone, respectively. %Activity was calculated from the signal intensity of DOX- cells, which was divided by the signal intensity observed for each indicated condition. Triplicate wells were assayed and the data represent the mean ± SD. (b) AKT/PKB activity downstream of PI3K signaling modified by the PCDH24 and/or the galectins. HCT116-PCDH24-HaloTag and HCT116 cells were cultured with or without DOX, transiently-expressed HaloTag-fused galectin-1 and galectin3. Western blot analysis of AKT/PKB in the cells was performed using antibodies against total AKT or phosphorylated AKT. Halo-Tag-fusion proteins were fluorescently-labeled using TMR HaloTag® ligand and detected by a FluoroImager FLA-3000 (Fujifilm). As a control, the PI3K inhibitor LY294002 was used. HCT116 cells were exposed to LY294002 (10 µM and 20 µM) for 24 h before the assay. (c) Subcellular localization of β-catenin in HCT116 cells treated with LY294002. Immunofluorescent images were obtained using an anti β-catenin antibody. (d) Proposed model for the mechanism by which PCDH24 regulates PI3K activation via galectin-1 and galectin-3 in colon cancer cells. In parental HCT116 cells (left), galectin-1 and -3 activate PI3K. The activation of PI3K by galectins activates AKT/PKB and also leads to the nuclear localization of β-catenin. Conversely, in HCT116 cells expressing PCDH24 (right), galectin-1 and -3 are trapped by PCDH24 at the cell membrane, and thus PI3K and AKT/PKB are not activated by the galectins. Subsequently, β-catenin is localized to the cell membrane and is prevented from activating the transcription of its target genes in the nucleus.