SOX2 Controls Fate of Brain Tumor Cells Figure 1. Ectopic elevation of SOX2 in U87 glioblastoma cells. (A) Photomicrographs of U87 cells infected with FUW-M2rtTA or FUW-M2rtTA and FUW-tetO-SOX2 lentiviruses. Photomicrographs were taken 48 hours after the addition of Dox to the culture medium, which induces ectopic SOX2 expression. (B) MTT assay of U87 glioblastoma cells, infected with FUW-M2rtTA or FUW-M2rtTA and FUW-tetO-SOX2 lentiviruses, cultured in various concentrations of Dox for 48 hours. Triplicates of each condition tested were averaged, and the error bars represent standard deviations. MTT values of cells cultured without Dox were set to one. This experiment was repeated two additional times, and similar results were obtained in each case. ‘*’ and ‘**’ indicate statistical significance (p,0.01 and p,0.001, respectively, student’s t-test). (C) Western blot analysis of SOX2 protein levels in nuclear extracts from U87 cells infected with FUW-M2rtTA and FUW-tetO-SOX2 lentiviruses, cultured without or with Dox for 24 hours to induce SOX2 expression. doi:10.1371/journal.pone.0044087.g001 SOX2 induction observed in our brain tumor studies presented above. In contrast to the previous report [18], we observed a notable reduction in cell number by light microscopy, 48 hours after SOX2 induction, in both MCF7 (Figure 4) and MDA-231 (Figure S2) breast cancer cells. Moreover, we observed a dose- dependent reduction in the cell population after the induction of SOX2 expression, demonstrated by MTT assay, in both MCF7 (Figure 4) and MDA-231 (Figure S2). Thus, the impairment of tumor cell growth following elevation of SOX2 is not unique to brain tumor cells. Moreover, the different conclusions reached here and in the former report [18] likely arise from differences in experimental design, as discussed more fully later in this report. Elevating SOX2 Levels in DAOY Cells Modulates the Expression of the Cell Surface Marker CD133 Cell lines, like in vivo tumors, are composed of a heterogeneous population of cells. To determine whether SOX2 may affect subpopulations within the DAOY cell line differently, we initially examined the surface expression of CD133 and SSEA1 with and without the elevation of SOX2. CD133 has been implicated in a number of cancers as a potential marker for identifying tumor- initiating cells, which are capable of reforming the original histopathology of the tumor [10,27,28]. SSEA1 has also been shown to identify a tumor-initiating cell population in brain tumors [29]. To examine the distribution of CD133+ and/or SSEA1+ cells following the elevation of SOX2, DAOY cells were transduced with both the FUW-M2rtTA and FUW-tetO-SOX2 lentiviral vectors. After transduction, the cells were exposed to 1 mg/mL of Dox for 24 hours, and harvested for two-color flow analysis as described in Materials and Methods. Exogenous expression of SOX2 caused an expansion of CD133+ and SSEA1+ populations, ,2.5-fold and 50%, respectively (Figure 5). However, it was unclear whether this increase in the proportion of CD133+ and SSEA1+ cells was due to deleterious effects of SOX2 on CD1332/SSEA12 cells, or to induction of CD133/SSEA1 expression. PLOS ONE | www.plosone.org 4 August 2012 | Volume 7 | Issue 8 | e44087