[5] were down regulated in the SOX2 knock down cells compared with MOCK controls (Figure 1, A, B). Quantitative real-time PCR (with primers provided in table S1) confirmed the observed changes of the array data (Figure 1, A, C, B). These observations made us to hypothesize that SOX2 is involved in the EMT process. We then repeated the experiment (Figure 1C) and found that the colorectal cancer cell with the MOCK control (SW620- MOCK) cells maintained mesenchymal cell morphology with spindle and fibroblastoid shape cells, while the cells with SOX2 knock down (SW620-shRNA-SOX2) showed epithelial cell morphology with epithelioid spreading cells (Figure 1D). These changes are typical changes during the MET process [3,5]. We then assessed the expression of classical genes involved in the EMT process including vimentin (a mesenchymal marker), E- cadherin and ß-catenin [5] by western blot and immunofluores- cence. We found that the expression of vimentin was almost completely abolished while the expression of E-cadherin was elevated significantly when SOX2 was knocked down (Figure 2A). Cell staining with SOX2 antibody confirmed the expression changes in E-cadherin and vimentin (Figure 2B). These results suggested that SOX2 is involved in the EMT process in colorectal cancer cells. SOX2 in Mesenchymal-Epithelial Transition We next examined, by RT-PCR, the mRNA level of the core EMT regulatory factors Snail, Slug, Twist, Zeb1 and Zeb2 [3] and found that the mRNA expression of Snail, Slug and Zeb1 were significantly downregulated in SW620shRNA-SOX2 cells compared with MOCK cells (Figure 2C). The expression of Zeb2 and Twist were not detectable in either cell lines (Figure 2C). Additionally, we analyzed the effect of SOX2 known down on the expression of other SOX2 and TCF family members using the microarray data that we generated previously [20], and found that SOX8 and TCF3 was also down regulated by about 5.6 and 2 fold respectively after SOX2 knock down compared with the mock control (Table S2). However, the expression level of TCF2 was up regulated by about 2.2 fold after SOX2 knock down comparing with the mock control (Table S2). To investigate whether overexpression of SOX2 could reverse the mesenchymal-epithelial transition (MET) process produced by SOX2 silencing, we overexpressed SOX2 as a SOX2-GFP construct in a SOX2 negative SW480 CRC cells. We confirmed by Western blot analysis that we were able to overexpress the SOX2 gene in SW480 cells (Figure S1A). RT-PCR analysis of the two markers of the EMT process showed that the E-cadherin expression did not change significantly (Figure S1B), but the expression of vimentin, the marker for the mesenchymal cells, Figure 1. The expression of key genes related to the EMT process and the cell morphology changed in response to SOX2 knock down. (A) A microarray analysis of the expression changes of genes related to epithelial and mesenchymal cells between SOX2 knock down and mock knock down SW620 colorectal cancer cells. (B) Quantitative real time PCR validation of expression changes in (A). GAPDH was used for normalization. The data are reported as mean 6 SEM. (C) Western blot analysis of the expression of SOX2 in SW620-mock and SW620-shRNA-SOX2 cells. (D) Cell morphology changes between SW620-mock cells and SW620-shRNA-SOX2 cells. The SW620-mock cells maintained a spindle and fibroblastoid morphology while the SW620-shRNA-SOX2 cell showed an epithelioid spreading morphology. Magnifications: the top panel, 1006; the middle panel, 2006; the bottom panel: 4006. doi:10.1371/journal.pone.0041335.g001 PLOS ONE | www.plosone.org 3 August 2012 | Volume 7 | Issue 8 | e41335