Figure 6

A PI3K inhibitor suppressed Akt phosphorylation and malignant properties of the transfectant cells. (A) Cells were cultured in serum free-medium overnight, and then treated with 50 μM LY294002 for 30 min. After treatment with serum-containing D-MEM for 5 min, cell lysates were prepared as described in “Materials and Methods”. Cropped blots were used here and uncropped imagesof blots are shown in Supplemental Figure S7 respectively. (B) To compare cell proliferation, MTT assay and ATP assay were performed. For MTT assay, transfectant cells and control cells (5.0 × 10³ cells/well) were prepared in 48-well plates in serum-containing D-MEM and cultured for 5 days (a). For ATP assay, transfectant cells and control cells (7.5 × 10² cells/well) were prepared in 96-well plates in serum-containing D-MEM and cultured for 4 days (b). Bars indicate mean ± S.D. (n = 3). (C) For invasion assay, transfectant cells and control cells (2.5 × 10⁴ cells/well) were cultured in serum-containing D-MEM for 16 h. Bars indicate mean ± S.D. (n = 3). (D) For adhesion assay to laminin, the transfectant cells and control cells (1.0 × 10⁴ cells) were treated with 50 μM LY294002 for 22 h and seeded in the wells of e-plates. Red and yellow lines mean transfectant cells, and green and blue lines mean control cells.