Adiponectin and Breast Cancer Figure 7. Hyperactivation of Akt/GSK3beta/beta-catenin signaling in adiponectin haplodeficient tumors. A, Components of the PI3K/ Akt/beta-catenin axis were characterized in the tumor cell lysates by Western blotting (upper panel) and nuclear beta-catenin activities analyzed using a TOPflash/FOPflash luciferase reporter assay (bottom panel). Results were expressed as fold changes relative to the values of samples derived from PyVT(+/2)/ADN(+/+) cells. #, P,0.01 vs PyVT(+/2)/ADN(+/+) group (n = 6). B, Various pharmacological inhibitors, including LY294002 for PI3K, PIK-75 for p110alpha, TGX221 for p110beta and IC8714 for p110delta, were used for the treatment of PyVT(+/2)/ADN(+/2) tumor cells at the concentration of 1026 M. The phosphorylations of Akt (pAkt), GSK3beta (pGSK3beta), and beta-catenin (pBeta-catenin), as well as their total levels in the cell samples treated with each specific inhibitor for 30 min were analyzed by Western Blotting (upper panel). After 24 hr incubation, the nuclear beta-catenin activities were evaluated using the TOPflash/FOPflash reporter assay (bottom panel). *, P,0.01 vs vehicle (n = 4). C, Primary tumor cells isolated from PyVT(+/2)/ADN(+/2) mice were cultured and treated without (vehicle) or with 1026 M of specific inhibitor of Akt-1/Akt-2 isoforms (Akti- 1/2) for 24 hr. Protein levels of phosphorylated Akt (pAkt), beta-catenin, and cyclinD1 in the cell lysates were analyzed by Western Blotting (upper panel) and the nuclear beta-catenin activities measured using a TOPflash/FOPflash luciferase reporter system (bottom panel). *, P,0.01 vs vehicle control (n = 3). D, Evaluation of the effects of various inhibitors on cell proliferation by [3H]-thymidine incorporation assay. CPM, counts per minute. *, P,0.01 vs vehicle in each treatment group (n = 5). Results were derived from three independent experiments. doi:10.1371/journal.pone.0004968.g007 Trx/TrxR redox system, and an altered Trx/TrxR redox balance could play important roles in promoting tumorigenesis in PyVT(+/ 2)/ADN(+/2) mice. In this study, we have shown that inhibition of both PI3K and Akt results in a reduced nuclear beta-catenin activities and protein stabilities, supporting the involvement of PI3K/Akt pathway in the PLoS ONE | www.plosone.org 9 March 2009 | Volume 4 | Issue 3 | e4968