Zhang et al. BMC Cancer 2011, 11:32 http://www.biomedcentral.com/1471-2407/11/32 Page 8 of 12 Figure 3 Tumor cell migration and invasion assays. All experiments were performed in triplicate and repeated twice. A, Migration assay. The RRIG1 sense and antisense-transfected MDA-MB-231 and MDA-MB-435 cells, respectively, were grown in G418-containing medium and then subjected to cell migration assay in Boyden chambers without Matrigel for 24 h. The cells that migrated were stained with 1% crystal violet solution and then counted and summarized. B, Invasion assay. These gene-transfected breast cancer cells were grown in G418-containing medium and then subjected to the cell invasion assay in Boyden chambers with Matrigel for 48 h. The invaded cells were stained with 1% crystal violet solution and then counted and summarized. *p < 0.05 vs. the control. important role in cell survival, and activation of Akt has been shown to overcome cell cycle arrest in G1 and G2 phases of the cell cycle to enable cell proliferation [19-22]. Nevertheless, knockdown of RRIG1 expression has had opposite effects in the regulation of breast cancer cell growth, colony formation, migration, and invasion. Interestingly, these effects were through RRIG1 regula- tion of some other gene expressions, such as phosphory- lated Stat3 and Rb. A previous study has shown the retinoic acid suppressed Stat3 phosphorylation in skin cancers [23]. Stat3 is a transcriptional factor and med- iates expression of a variety of genes in response to cell stimuli and thus plays a key role in many cellular pro- cesses such as cell growth and apoptosis [24,25]. RRIG1 protein contains two putative Src homology 3 (SH3) domain-binding motifs that were characterized in a previous study [26], and our preliminary data showed that RRIG1 was able to bind to Src protein [8]. Previous studies [see reviews in ref. [25]] demonstrated that the role of STAT3 as a downstream signal transducer in Src family kinase-mediated tumorigenesis suggests that RRIG1 may suppress Stat3 activity through binding to Src protein. However, this needs further investigation. Furthermore, our current study also confirmed our previous finding that RRIG1 inhibits RhoA activation to execute some of its biological functions [5]. RhoA is a small GTPase protein known to regulate the actin cytos- keleton in controlling cell mobility and growth and modulating gene expression [27-31]. The aberrant acti- vation of RhoA proteins was found to cause cell growth, transformation, invasion, and metastasis in experimental models of carcinogenesis, and inhibition of RhoA