PC3, 22RV1, LNCaP, and DU-145 cells were obtained from American Type Culture Collection (ATCC, Manassas, VA). These cells were maintained in culture medium, according to the manufacturer's instructions.
pMig-Slug was constructed by cloning human SLUG gene into pMIGR1 retroviral vector. pLKO.1-Slug shRNA1 (target sequence: 5'-CAGCTGTAAATACTGTGACAA-3'), pLKO.1-Slug shRNA2 (target sequence: 5'- CCAAATCATTTCAACTGAAA-3'), pLKO.1-CXCL12 shRNA1 (target sequence: 5'-TGTGCATTGACCCGAAGCTAA), and pLKO.1-CXCL12 shRNA2 (target sequence: 5'-GCCAACGTCAAGCATCTCAAA-3') were obtained from Open Biosystem (Huntville, AL). pLKO.1 control shRNA (containing non-target scramble shRNA, Addgene plasmid #1864) were purchased from Addgene (Cambridge, MA).
Viral Production and Infection
293T cells were seeded at 3 × 105 cells per well in a 6-well plate. The next day, a mixture of plasmid DNA was transfected separately into 293T cells using Superfect transfection reagent (Qiagen, Valencia, CA). For retrovirus production, pCL-Ampho (packaging plasmid) was mixed with pMig-based retroviral vectors. To generate the lentiviruses, the packaging plasmids (pCMV-VSVG and psPAX2) were co-transfected with pLKO.1-Slug shRNA or pLKO.1-control shRNA (containing non-target shRNA). The viruses were collected 24 hr after transfection. For viral infection, PC3, 22RV1, or DU-145 cells were seeded at 50% confluence in 6-well plates. The next day, the virus-containing supernatants from 293T cultures were mixed with polybrene (Sigma, St. Louis, MO) at a final concentration of 4 mg/ml, and added to the cells in each well. The plate was centrifuged at 2,000 rpm for 1 hr at 35°C, and returned to the cell culture incubator. PC3, 22RV1, DU145, and LNCaP cells were infected with retroviruses (pMig-Slug or pMig vector) for 3 times to achieve 100% transduction in these cells. Cells infected with pLKO.1 lentiviruses were selected with puromycin (1 μg/ml), starting at 48 hr after infection.
RNA Isolation, cDNA Synthesis, RT-PCR, and qPCR Analysis
Total RNA extraction from cultured cells was accomplished by using RNeasy Plus mini kit (Qiagen, Valencia, CA). cDNA was synthesized by random priming from 1 μg of total RNA with the SuperScript III First-Strand Synthesis Super Mix kit (Invitrogen, San Diego, CA), according to the manufacturer's protocol. Primers used for the RT-PCR and qPCR analysis were synthesized by Integrated DNA Technologies (Coralville, IA). RT-PCR was performed by using the Hotstar Taq DNA polymerase kit (McLab, San Francisco, CA), and qPCR was performed by using the Perfecta SYBR Green FastMix (Quanta Bioscience, CA), according to the manufacturer's protocol. Data were analyzed by using the comparative CT method; CT refers to the ''threshold cycle,'' and is determined for each experiment using MyiQ software. Quantities of gene specific mRNA expression were determined by the CT method. Amplification of GAPDH was performed for each reverse-transcribed sample as an endogenous quantification standard. The fold-difference in gene expression was determined by 2_ΔΔCT. ΔΔCT is equal to (ΔCT of experimental conditions -ΔCT of control conditions). ΔCT is equal to (gene-specific CT -GAPDH CT). The primers are as following: SLUG, 5'-CTTCCTGGTCAAGAAGCA-3' and 5'-GGGAAATAATCACTGTATGTGTG-3'; CXCR4, 5'-ATATACACTTCAGATAACTACACCGAG-3' and 5'-TCAGTTTCTTCTGGTAACCCATGACCA-3'; CXCL12, 5'-ACCGCGCTCTGCCTCAGCGACGGGAAG-3' and 5' TGTTGTTCTTCAGCCGGGCTACAATCTG-3'; MMP9,5'-AGCGGGCGGCGCCTCTGGAGGTTCGA-3' and 5' CCTGGCAGAAATAGGCTTTCTCTCGGT-3'; GAPDH, 5' ATTGACCTCAACTACATGGTTTACATG-3' and 5'-TTGGAGGGATCTCGCTCCTGGAAG-3'.
Enzyme-linked Immunosorbent Assay (ELISA)
Conditioned cell culture medium was centrifuged and an SDF1-α immunoassay kit (R&D Systems Inc. Minneapolis, MN) was used for CXCL12 detection. 100 μl of sample or control (or standard) was added into each well, according to the manufacturer's protocol. The optical density of each well was measured within 30 min, using a microplate reader set to 450 nm.
Western Blot Analysis
The cells were lysed in the protein lysis buffer (20 mM Tris, 100 mM NaCl, 1 mM EDTA, 0.5% Triton X-100, 1 mM beta glycerophosphate, 1 mM sodium orthovanadate), supplemented with 1 ml protease inhibitor cocktail (Sigma, St. Louis, MO). The protein samples were analyzed by Western blot analysis using an ECL kit (Pierce, Rockford, IL) with antibodies against following antigens: Slug (ANASPEC, Fremont, CA), CXCR4 (Abcam, Boston, MA), GAPDH (Bethyl Laborotaries, Montgomery, TX).
Zymographic Analysis of MMP activity
Cells overexpressing pMig or pMig-Slug (70-80% confluence) were washed twice with PBS, and the medium was changed to serum free cell culture medium. After 48 hr, the conditioned medium was collected and centrifuged for 5 min at 400 × g. A 500 μl aliquot was concentrated to < 100 ul in a Microcon concentrator (Millipore, Billerica, MA) at 6500 × g at 4°C. Protein concentration was determined using BCA assay (Thermo Scientific, Rockford, IL), and 20 μg of the protein from each sample was electrophoresed on a 10% zymography gel containing 0.1% gelatin (Invitrogen, San Diego, CA). MMP activity was detected by incubating the gel in 1× Zymogram Renaturing Buffer for 30 min at room temperature and then equilibrating the gel for 30 min at room temperature with gentle agitation. The gel was incubated with fresh 1× Zymogram Developing Buffer overnight, followed by staining with Coomassie Blue for 30 min. Contrast was adjusted by destaining with Coomassie destaining solution (Methanol: Acetic acid: Water (50: 10: 40). The staining gels were then air-dried in cellophane mounts and images were captured.
Wound Healing Assay
The cells were seeded in a 12-well plate (15 × 104). After the cells formed a confluent mono layer, scratches were performed using a 100 ul tip. The culture medium was replaced with fresh complete medium. The closure of scratch was analyzed under the microscope and images were captured at 18 - 24 hr after incubation.
The cells were seeded at 6 × 104 cells per well into the 96-well plate of an Oris™ Cell Invasion Assay Kit (Platypus, Madison, WI). The plate was incubated for ~16 hr at 37°C. The stoppers were then removed. Collagen I Overlay was added to create a 3-D ECM environment for invasion and incubated for 1 hr at 37°C. Cell culture medium was added and the cells were allowed to invade for 72 hr, and were stained with DAPI before images were captured.
qPCR data and cell growth data were analyzed by the Student's t-test (one-tailed). P < 0.05 was used to define statistically significant differences.