Zhang et al. BMC Cancer 2011, 11:32 http://www.biomedcentral.com/1471-2407/11/32 the percentage of control was calculated using the for- mula: value of the RRIG1 sense or antisense transfected cells/value of the vector-only transfected cells. RhoA activation assay The RRIG1 sense and antisense cDNA-transfected breast cancer cells were detached with 0.05% trypsin, counted, reseeded, and cultured in new dishes in DMEM with 10% FCS for 16 hours and then in DMEM without FCS for an additional 12 hours. To activate RhoA, the cells were cultured in DMEM with 10% FCS for 6 h, and the total cellular protein was extracted in an ice-cold lysis buffer containing 20 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyropho- sphate, 1 mM b-glycerophosphate, 1 mM Na3VO4, and 1 μg/ml leupeptin. Activated GTP-bound Rho protein in the cell lysates was pulled down using a recombinant GST-tagged Rhotekin Rho-binding domain (Millipore, Billerica, MA) and analyzed in western blots using anti- RhoA antibody (Santa Cruz Biotechnology). Levels of the activated RhoA protein were normalized with the total cell lysates that were not subjected to the pulldown assay. Results Immunohistochemical analysis of RRIG1 expression in breast tissue specimens In this study, we first verified the specificity of our rab- bit polyclonal anti-RRIG1 antibody using three different experiments. Briefly, we first performed western blot with this antibody to detect expression of RRIG1 protein in vitro translated with an in vitro translation kit from Promega (Madison, WI). The data showed that this anti- body specifically recognized RRIG1 protein (Additional Figure S1a). Meanwhile, in two different cancer cell lines we detected RRIG1 expression, which had been established by Northern blot in our previous study. We found that RRIG1-positive esophageal cancer cell line TE-8S22 expressed RRIG1 protein, whereas the PC3 cells did not show the target band, although the western blot showed a number of nonspecific bands (Additional Figure S1b), indicating that this antibody is not suitable for western blot assay. To determine whether this antibody could be used for immunohistochemistry, we compared the data between immunohistochemistry and in situ hybridization for expression of RRIG1 mRNA and protein in the matched cells and tissues (Additional Figure S1cd). These data suggest that this antibody is suitable for immunohisto- chemistry. Next, we used immunohistochemical analysis to determine the RRIG1 expression in breast tissue spe- cimens and found that RRIG1 was expressed in 14 of 15 normal mammary glands, 8 of 9 cases of atypical Page 4 of 12 hyperplasia of the mammary gland, 6 of 10 ductal carci- noma in situ tissues, and 50 of 77 invasive breast cancer tissues (P = 0.023 between normal and invasive cancer by Fisher exact test) (Figure 1). Finally, we associated RRIG1 expression with the clini- copathologic features of the breast cancer patients and found a statistically significant reverse correlation between RRIG1 expression and lymph node metastasis in breast cancer (Table 1). RRIG1 regulation of breast cancer cell growth, migration, and invasion We then detected expression of RRIG1 mRNA in 6 dif- ferent breast cancer cell lines using semiquantitative RT- PCR and found that MDA-MB-435 and T47D expressed high levels of RRIG1 mRNA and that MCF-7 and SK-Br3 expressed low levels of RRIG1 mRNA. In contrast, MDA-MB-231 and ZR75-1 did not express RRIG1 mRNA (Figure 2A). Given these findings, we chose MDA-MB-231 and MDA-MB-435 for modulation of RRIG1 expression to assess the changed cell behaviors and gene expression. We transiently transfected RRIG1 sense and antisense cDNAs into the MDA-MB-231 and MDA-MB-435 cell lines, respectively. After gene trans- fection, RRIG1 expression was knocked down by RRIG1 antisense vector in MDA-MB-435 cells, while RRIG1 cDNA transfection restored RRIG1 expression in MDA- MB-231 cells (Figure 2B). Next, we used the MTT assay to detect the changed cell viability by RRIG1 and found that restoration of RRIG1 expression reduced cell viabi- lity in MDA-MB-231 cells, but knockdown of RRIG1 expression induced cell viability in MDA-MB-435 cells after 5 d of cultures (Figure 2C). Furthermore, RRIG1 expression decreased the number of colonies in MDA- MB-231 cells, and knockdown of RRIG1 expression in MDA-MB-435 cells increased the numbers of colonies in soft agar (Figure 2D). In addition, our data also showed that RRIG1 decreased migration and invasion capacity of MDA-MB-231 cells but increased the migratory and invasion capacity of MDA-MB-435 cells (Figure 3). RRIG1 regulation of gene expression in breast cancer cells To determine the underlying molecular events of RRIG1 in the inhibition of breast cancer cell proliferation, migra- tion and invasion, we performed semiquantitative RT-PCR and western blotting analysis to assess RRIG1 regulation of gene expression and the RhoA activation assay to detect the changed RhoA activity. We found that RRIG1 expres- sion downregulated p-Erk1/2, p-AKT, total AKT, c-Jun, and MMP9 expression in MDA-MB-231 cells, whereas knockdown of RRIG1 expression upregulated p-AKT, total AKT, p-Stat3, p-RB, and E2F-1 expression in MDA- MBN-435 cells (Figure 4). However, these data demon- strated that RRIG1 sense and antisense cDNAs regulated