Zhang et al. BMC Cancer 2011, 11:32 http://www.biomedcentral.com/1471-2407/11/32 described previously [6,7]. After preliminary investiga- tion to determine the optimal number of PCR cycles (between 25 and 34), we used 32 cycles of PCR to amplify RRIG1 mRNA. GAPDH was used as a loading control. The primers for RRIG1 mRNA were 5’- CTCCCAGGGTGCCATATTT-3’ and 5’-GTCATA- GAGCACCCGAGCTT-3’, which generated a 211-bp band. The primers for MMP-9 expression were 5’- GCACGACGTCTTCCAGTACC-3’ and 5’-GTTTGTAT CCGGCAAACTGG-3’, which generated a 224-bp band. The primers for SH3GLB2 expression were 5’-GCAGA- CAGCACCAAGAACTG-3’ and 5’-TTTTCAGCTT CTGCCACCTT-3’, which generated a 233-bp band. GAPDH primers were 5’-CCCTTCATTGACCTCAAC- TACATGG-3’ and 5’-CATGGTGGTGAAGACGCCAG- 3’, which generated a 192-bp band. MTT assay The cells were grown and transiently transfected with RRIG1 sense or antisense cDNA and then grown in G418-containing medium for 1 or 5 days. For the methyl thiazolyl tetrazolium (MTT) assay, 20 μL of MTT (5 mg/ml, Sigma, St. Louis, MO) was added to each well of the 96-well plates and incubated for an additional 4 h. After the growth medium was removed, 100 μL of dimethyl sulfoxide was added to the wells to dissolve the MTT crystal, and the optical densities were measured with an automated spectrophotometric plate reader at a single wavelength of 540 nm. The percentage of cell growth was calculated using the formula: % con- trol = ODt/ODc × 100, where ODt and ODc are the optical densities for transfected and vector control cells, respectively. Colony formation assay The ability of cells to form colonies in soft agarose is indicative of anchorage independence and is used as an in vitro criterion of transformation. We examined whether these cells can form colonies in soft agar after gene transfection [9]. Briefly, 2 × 103 gene-transfected cells were mixed in low-temperature-melting agarose (0.35%) and then placed on top of solidified agarose in 60-mm-diameter dishes. After the top agarose with cells solidified in the cold room for 15 min, the dishes were incubated in a humidified atmosphere of 95% air and 5% CO2 at 37°C for 21 days. At the end of the experi- ments, the colonies were visualized by incubation with MTT at 37°C for 4 h and counted under an inverted microscope at 40× magnification. Migration and invasion assay Boyden chambers coated with and without Matrigel (obtained from BD Biosciences, Bedford, MA) were used for assaying tumor cell invasion and migration ability, Page 3 of 12 respectively [5]. The transfected cells were first starved in medium without fetal calf serum (FCS) overnight, and the cells (5 × 104) were resuspended in the FCS- free medium and put into the top chambers in triplicate. The lower chamber was filled with 10% FCS as the che- moattractant and incubated for approximately 24 h for the migration assay and 48 h for the invasion assay. The surface was then wiped with a cotton swab to remove the cells on the upper surface. The cells that invaded the Matrigel and attached to the lower surface of the fil- ter were fixed and stained with 1% crystal violet solu- tion. The membranes with or without Matrigel were then gently removed from the chamber and mounted on glass slides. Six microscopic fields (at 100× magnifi- cation) per chamber were photographed. The cells in the photographs were then counted, and the data were summarized as means ± standard deviation and pre- sented as the percentage of control. Student t-test was used to determine statistical differences between the control and RRIG1 sense- or antisense-transfected breast cancer cells. Protein extraction and western blotting Total cellular protein was isolated from the gene-trans- fected breast cancer cells as described previously [5-7]. The protein concentration was then measured with a Bio-Rad Protein Assay Kit II (BioRad Laboratories, Her- cules, CA) according to the manufacturer’s protocol. Samples containing 50 μg of protein from control or treated cells were separated by 10-14% polyacylamide sodium dodecyl sulfate-polyacrylamide gel electrophor- esis gels and then transferred electrophoretically to a Hybond-C nitrocellulose membrane (GE-Healthcare, Arlington Heights, IL) at 500 mA for 2 h at 4°C. The membrane was subsequently stained with 0.5% Ponceau S (Sigma) containing 1% acetic acid to confirm that pro- teins were loaded equally and to verify transfer effi- ciency. The membranes were next incubated overnight in a blocking solution containing 5% bovine skim milk and 0.1% Tween 20 in PBS at 4°C. The next day, the membranes were incubated with primary antibodies for 2 h at room temperature. The antibodies used were anti-p-Erk1/2, t-Erk1/2, p-Akt, t-Akt, p-Stat3, t-Stat3, p- Rb, and Rb (Cell Signaling Technology, Beverly, MA); anti-c-Jun and E2F-1 (Santa Cruz Biotechnology, Santa Cruz, CA); and anti-b-actin (Sigma). The membranes were washed in PBS and incubated for 1.5 h with horse- anti-mouse or goat-anti-rabbit secondary antibody (GE- Healthcare) diluted 1:5000. The membranes were then incubated with enhanced chemiluminescence solution (GE-Healthcare) for 1-2 min and exposed to X-ray film. The target band of proteins from western blots was quantified by using NIH ImageJ 1.34s software (NIH, Bethesda, MD) and normalized to b-actin. After that,