1C). Together, these results indicate that

AIB1 is important for CCA cell proliferation. To explore the mechanism by which knockdown of AIB1 inhibits proliferation of CCA cells, cell cycle analysis was performed to examine whether AIB1-knockdown cells are arrested in a specific phase of the cell cycle. Cells were synchronized by serum starvation for 24 hours, then the cell cycle was measured by flow cytometric analysis after serum addition for 24 hours. As shown in Fig. 2A, AIB1 knockdown induced G2/M arrest in QBC939 cells. To www.selleckchem.com/screening/autophagy-signaling-compound-library.html better understand the role of AIB1 in cell cycle progression, cells were synchronized to the G1/S boundary by the double thymidine block method or arrested at G2/M phase by thymidine/nocodazole, then the cell cycle was measured by flow cytometric analysis after drug withdrawal. The results in Supporting Fig. 2 confirmed the conclusion that down-regulation of AIB1 resulted in G2/M arrest. In addition, the subG1 population of AIB1-knockdown cells, which represents apoptotic cells, was significantly larger than that of control cells, indicating that AIB1 protein is required for both proliferation and survival of CCA cells. To address the molecular mechanism underlying the crucial role of AIB1 in control of the CCA cell cycle, we examined

mTOR inhibitor the effect of knocking down AIB1 on key G2/M regulatory genes expression by western blotting. The expression of G2/M regulatory proteins such as Cyclin A, Cyclin B, Cdk1, and Cdc25 was detected at different time after serum addition following synchronization of cells by serum starvation for 24 hours. The results showed that the expression of mitosis-promoting factors including Cyclin A, Cyclin B, and Cdk1 recovered more slowly and less in AIB1-knockdown QBC939 cells than those in control cells, whereas the expression of G1-related factor Cyclin D1 was comparable in both cells (Fig. 2B). In addition, down-regulation of AIB1 markedly inhibited the expression of phosphorylated Akt (Fig. 2B). Because activation of the Akt

signaling pathway can lead to the promotion of cell cycle progression at the G2/M phase,7, 8 we investigated whether reduced Akt signaling pathway is involved in G2/M arrest by see more using the PI3K/Akt inhibitor LY294002 to treat QBC939 cells. As shown in Fig. 2C, LY294002 treatment reduced the phosphorylation of Akt and inhibited the recovery of Cyclin A, Cyclin B, and Cdk1 after serum addition. These results indicate that down-regulation of AIB1 inhibits the cell cycle progression at least in part through reducing the activity of Akt signaling pathway. Resistance of CCA to chemotherapeutic drugs is a major problem that limits the effectiveness of chemotherapies used to treat this cancer. Better understanding drug sensitivity and the mechanism of drug resistance of this cancer is essential.