Inhibition of the PI3-K/Akt and mTOR/p70^S6K signaling pathways induces G₀/G₁ cell-cycle arrest, up-regulates p27^Kip1 inhibitor, and down-regulates Skp2 and cyclin D expression. (A) Granta 519 and SP-53 cells were cultured in presence of solvent alone (“C”), 50 μM LY294002 (“LY”), 20 μM SH-5, or 0.1 μM rapamycin (“R”) for 24 hours. Cell cycle was analyzed by FACS after DNA staining with PI in cells permeabilized with 0.01% NP40. (B) Granta 519, SP-53, and Jeko-1 cells were treated with solvent alone, 50 μM LY294002 (LY), or 0.1 μM rapamycin (R) for 24 hours. Total-cell lysates corresponding to 100 μg of proteins were analyzed by immunoblotting for the indicated proteins. Anti-pRb Ab recognizes the hyperphosphorylated (**) and hypophosphorylated (*) Rb proteins. (C) Granta 519 cells were treated with solvent alone (DMSO) or 50 μM LY294002 (LY) for 24 hours, and 100 μg of nuclear (N) and cytoplasmic (C) fractions were analyzed by immunoblotting for the indicated proteins. Localization of nucleus-specific protein topoisomerase IIα (Topo IIα) and cytoplasm-specific tubulin was carried out to confirm the purity of the nuclear and cytoplasmic fractions.