SW620shRNA-SOX2, were cultured in RMPI-1640 medium with 10% fetal bovine serum. Immunofluorescence cell staining Immunofluorescence cell staining was performed using the following primary antibodies: Rabbit anti-SOX2 (Epitomics), 1:250; mouse anti-Vimentin (Boster), 1:100; mouse anti-E- cadherin (Abcam), 1:100; and rabbit anti-beta-catenin (Epitomics), 1:250. Cells were seeded on the cover slips and incubated for 24 hours at room temperature, and then fixed with formalin for 20 min, washed with PBS and blocked with PBS containing 1% of BSA and 0.25% Triton X-100. Slides were incubated with primary antibody overnight at 4uC, washed with PBS, and then incubated with secondary antibody conjugated with FITC (green) or Cy3 (red) (Millipore) for 1 hour. After washing, cover slips were attached to glass slides. Cells were imaged using a confocal microscope. Western Blot Analysis For whole-cell extract, cells were grown to 70% confluency, then washed with cold PBS buffer, and lysed on ice for 30 min in 200 ul RIPA buffer. Cells lysates were cleared by centrifuging at 14,000 rpm for 15 minutes. For nuclear extract, cells were lysed using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo SCIENTIFIC). Protein concentrations were estimated using Pierce BCA protein Assay Kit (Thermo SCIENTIFIC). 30 mg of proteins was denatured at 95u with loading buffer for 5 min and separated by electrophoresis in 12% SDS-PAGE gels for low molecular weight proteins. Proteins were transferred onto a PVDF membrane, blocked 1 hour in 5% slim milk in TBST. Primary antibodies were diluted to incubate with PVDF mem- brane overnight at 4u [anti-SOX2 (2683-1 Epitomics), 1:5000; anti-Vimentin (BM0135, Boster), 1:200; anti-E-cadherin (ab1416, Abcam), 1:1000; anti-GAPDH (ab9483, Abcam), 1:5000; anti- beta-catenin (1247-1, Epitomics), 1:5000; anti-Lamin B (sc- 365962, Santa cruz), 1:500; anti-alpha-tublin (2871-1, Epitomics), 1:5000; anti-TCF/LEF1, 1:1000 (2091-1, Epitomics)]. After washing, the membranes were incubated for 1 h at RT temperature with HRP linked secondary antibody [anti-rabbit (1:5000), anti-mouse (1:5000) (Abcam)], and signals were detected using ECL reagents (Thermo Scientific). RNA isolation and RT-PCR Total RNA was extracted after 24 hours postseeding with Trizol. 2 ug RNA for each analyzed sample was reverse transcribed in a final volume of 25 ml using M-MLV Reverse Transcriptase (Promega). Quantitative real-time PCR reaction was performed in a final volume of 20 ml containing 1 ml of cDNA (1:10 dilution) and 400 nM of primers (All primers were listed in supplementary table S1) and SybrGreen PCR premix (Takara), and run in a Biorad Cycler. All samples were amplified in triplicate using the following cycle scheme: 95uC for 2 min, 35 cycles of 95uC for 15 s, 60uC for 30 sec and 72uC for 20 sec. Final mRNA levels were normalized to GAPDH levels. All results are plotted as columns (mean) and bars (SD). Cell migration and invasion assays Cell migration assay was performed as described using Transwells (24-well Insent; 8 mM pore size polycarbonate membrane) obtained from Corning. Cells (56105) in 0.5 ml serum-free medium were placed in the upper chamber, whereas the lower chamber was loaded with 0.8 ml medium containing 10% FBS. The total number of cells that migrated into the lower SOX2 in Mesenchymal-Epithelial Transition chamber was counted after 60 h of incubation at 37uC with 5% CO2. Six visual fields were chosen for counting. The transwell cell invasion assay was performed using transwells that were pre- loaded with a layer of Matri-gel (Sigma-Aldrich) on the upper surface. The rest experiment procedure is same with cell migration assay. In vivo studies All animal experiments were approved by governmental authorities. Six-week old female nude mice were each injected via the tail vein with SW620shRNA-SOX2 or SW620mock (56106). Mice were sacrificed 30 days after the tail vein injection, and lung and liver were embedded in paraffin and sliced for HE staining to examine for metastasis. Gelatin zymography Cells were plated at a density of 106 in 6-well plates. After 16 hours, cells were washed with PBS and incubated in 300 ml of serum-free medium for 24 hours. 55 ml aliquots of conditioned medium were diluted in sample buffer (5% SDS), 20% glycerol in 0.4M Tris pH 6.8 containing 0.02% bromophenol blue without 2- mercaptoethanol and were loaded for zymography on a 10% polyacrylamide gel containing 1 mg/ml gelatin. Afterwards, gels were washed for 30 minutes in 2.5% Triton X-100 and incubated for 24 hours at 37uC in 50 mM Tris, pH 7.5, 10 mM CaCl2, 0.02% NaAzide. The buffer was decanted and the gels stained with 0.5% Coomassie blue G in 30% methanol and 10% acetic acid for 10 minutes at room temperature on a rotary shaker. Stain was washed out with water until clear bands were seen. Areas where proteolytic activity degraded the gelatin manifested as absence of staining. Immunohischemistry Paraffin-embedded tumors samples were obtained from 44 colorectal cancer patients with approval from the institutional ethics committee of the 2nd hospital of Zhejiang University Medical College. Immunohistochemical (IHC) analyses were performed on 3-mm, formalin-fixed and paraffin-embedded sections, after microwave-assisted antigen retrieval in 0.1 mol/L of citrate buffer (pH 6.0) and subsequent incubation with 3% hydrogen peroxide. Primary antibodies for SOX2 were diluted at 1:250 (Epitomics) and secondary antibody was used at 1:200 dilution for IHC. Stained sections were photographed using a fluorescence microscope. Statistical analysis The results are reported as mean 6 SD. Statistical analysis was performed using Student’s t-test or Chi-square test as appropriate. P,0.05 was considered to be statistically significant. Results Silencing SOX2 Promotes Mesenchymal-Epithelial Transition (MET) in Colorectal Cancer Cells The epithelial to mesenchymal transition (EMT) process is marked by changed in the expression of epithelial and mesenchy- mal markers. In a previous study, we successfully knocked down SOX2 in a colorectal cancer cell line SW620, in which SOX2 is highly expressed [20]. We found that a number of epithelial markers including TJP1, TJP2, TJP3, CGN and KLK10 [5] were up-regulated in response to SOX2 knock down compared to mock control cells using microarrays [20]. In addition, a series of mesenchymal marker genes including VIM, FN1, SNAI2, CLDN1 PLOS ONE | www.plosone.org 2 August 2012 | Volume 7 | Issue 8 | e41335