SOX2 Controls Fate of Brain Tumor Cells Figure 8. Effects of SOX2 overexpression on CD1332 DAOY cells. i-SOX2-DAOY cells were FACS sorted into CD1332 and CD133+ populations. SSEA1+ cells were excluded from both populations. (A) CD1332 cells were cultured in medium without or with Dox (0.5 mg/mL) for 24 hours. Cells were then collected for FACS analysis of surface expression of CD133 and SSEA1. (B) RNA was isolated from CD1332 cells cultured without and with Dox (0.5 mg/mL) for 24 hours. cDNA was prepared and RT-qPCR was conducted. Values represented are differences in Ct value (normalized as described in Figure 7), where a positive value indicates increased expression of a given transcript within the Dox supplemented population. (C) A scatter plot of transcript expression, as determined by Affymetrix microarray analysis, comparing i-SOX2-DAOY cells cultured in the absence or presence of Dox (0.5 mg/mL) for 24 hours. Transcript expression from CD1332 DAOY cells cultured without Dox is represented on the y- axis; whereas, transcript expression from cells cultured with Dox is represented on the x-axis. A single replicate of each condition was analyzed. RT- qPCR analysis (subfigure B) was used to validate the array data. (D) Gene ontology analysis comparing genes that were upregulated or genes that were downregulated 2-fold, in CD1332 DAOY cells cultured in the absence or presence of Dox. The y-axis represents the percentage of up- or downregulated transcripts that reside within various ontology categories. doi:10.1371/journal.pone.0044087.g008 SOX2 will provide significant insight into the biological processes that are required by cancers employing SOX2 for their tumorigenicity. Our findings with brain tumor cells raised an important question: do brain tumor cells respond differently to increases in SOX2 than other types of tumors? To address this question, we elevated SOX2 in prostate and breast cancer cells and observed that a 2- to 3-fold induction of SOX2 significantly reduced cell number in vitro (Figures 3 & 4). While our findings differ significantly from those reported by others [18,19], it is likely that these differences are attributable to important differences in experimental design. In our studies, SOX2 was elevated from an inducible promoter in the total population of infected DU-145 prostate cancer, as well as MCF7 and MDA-231 breast cancer cells. Remarkably, these studies demonstrate that increased SOX2 levels are detrimental to the majority of the cell population soon after SOX2 is increased. Conversely, others isolated and then studied tumor cells that stably express elevated levels of SOX2 (,3- to 4-fold) from a drug selected transgene. The differences observed in our studies and those of others appear to be due to the selection of a subpopulation of cells that not only tolerate elevated levels of SOX2, but exhibit enhanced proliferative capacity both PLOS ONE | www.plosone.org 12 August 2012 | Volume 7 | Issue 8 | e44087