As shown in Fig. 5A, as expected, expression of shRNA against Bcl-2 results in loss of protein Bcl-2 in both cytoplasm in nucleus, ectopic expression of Twist1 expression vector led to an increased expression of both cytoplasm and nucleus but predominantly in nucleus (Fig. 5A, right panel). However, when cells contain both Twist1 expression vector and

shRNA against Bcl-2, the nuclear expression of Twist1 is completely attenuated. To further demonstrate whether Bcl-2 facilitates the nuclear transport of Twist, we examined these cells in hypoxia conditions and in the presence of PI3K inhibitor overexpression of Bcl-2 rather than knockdown by shRNA. As shown in Fig. 5B, either hypoxia or ectopic expression of Bcl-2 can lead to up-regulation of expression of Twist1 with preferential expression in

the nucleus. These results further support the interaction between Bcl-2 and Twist1; Bcl-2 could be an important cofactor to facilitate the transport of Twist1 to the nucleus (Fig. 5A,B). To examine how interactions between Bcl-2 and Twist1 affect global gene expression, we examined the promoters bound to Twist1 using a ChIP-sequence analysis for HepG2-control, HepG2-Twist1, and HepG2-BT that are transfected with Inhibitor Library screening both Bcl2 and Twist1. The DNA fragments bound to Twist1 picked up by ChIP assay were sequenced. The results showed that the number of gene promoters that bound to Twist1 in the HepG2-BT expressing both Bcl-2 and Twist1 cells reached 100, whereas only 43 promoters were detected in HepG2 transfected with Twist1 expression vector alone (Fig. 6A). These genes are involved in many processes such as cell signal transduction, cell proliferation, angiogenesis, and cytoskeleton formation (detailed information is provided in the Supporting Materials, Table s7). To verify whether key signal transduction pathways were activated

by the interaction of Bcl-2/Twist1, reporter gene plasmids with AP1, Stat3, and NF-κB activation Carteolol HCl sequences were used in the HepG2-BT and control cells. The results showed that the AP1 and Stat3 activities in the HepG2-BT group significantly increased. In contrast, the NF-κB transcriptional activity did not significantly change compared with the control and HepG2-Twist1 groups (Fig. 6B). The western blot analysis also showed similar results; a high level of c-Jun, p-c-Jun, as well as Stat3 was observed in the HepG2 cells expressing both Bcl-2 and Twist expression vector (HepG2-BT). We also examined the global changes in mRNA for HepG2-control, HepG2-Bcl-2, HepG2-Twist1, and HepG2-BT using cDNA array. Cluster and comparative analyses showed a distinct pattern of mRNA expression when these cells exogenously expressed transfected Bcl-2, Twist1 or a combination of both (Supporting Fig. s3).