SOX2 Controls Fate of Brain Tumor Cells Figure 3. Ectopic elevation of SOX2 in DU-145 prostate cancer cells. (A) Photomicrographs of DU-145 prostate cancer cells infected with FUW-M2rtTA or FUW-M2rtTA and FUW-tetO-SOX2 lentiviruses, cultured in the presence of Dox, at the indicated concentration, for 48 hours. (B) MTT assay of DU-145 prostate cancer 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. ‘**’ indicates statistical significance (p,0.001, student’s t-test). (C) Western blot analysis of SOX2 protein levels in nuclear extracts from DU-145 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.g003 Annexin V staining, respectively, following 48 hours of ectopic SOX2 expression (data not shown). We also examined b- galactosidase activity, which is reported to increase when cells become senescent [30]; however, we did not detect increased b- galactosidase staining following ectopic SOX2 elevation (data not shown). Thus, i-SOX2-DAOY cells exited the cell cycle (Figure 6D), and likely became quiescent during the time period examined. Exogenous SOX2 Expression Slows the Long-term Growth of Brain Tumor Cells From previous reports demonstrating that elevation of SOX2 can enable certain tumor cells to grow more rapidly, we postulated that, given enough time in the presence of elevated SOX2, a subpopulation of tumor cells could emerge which expressed exogenous SOX2 and proliferates rapidly. To address this possibility, we used i-SOX2-DAOY cells to examine the long- term effects of SOX2 elevation on the growth of DAOY medulloblastoma cells. For this purpose, i-SOX2-DAOY cells, cultured in the absence and presence of Dox, were passaged at clonal density every week for four weeks. Although i-SOX2- DAOY cells were able to proliferate slowly and form small colonies, the growth of i-SOX2-DOAY cells remained low when SOX2 was elevated, even after 1 month in culture (Figure S4). To determine whether exogenous SOX2 expression could impair the long-term growth of other brain tumors types, we stably engineered U87 glioblastoma cells for Dox-inducible expression of SOX2 (i-SOX2-U87, Figure S5) using the same lentiviral vectors used to engineer i-SOX2-DAOY cells. We first characterized the short-term effects of SOX2 elevation on i-SOX2-U87 cells. Similar to our initial observations in U87 cells (Figure 1), elevation of SOX2 in i-SOX2-U87 cells (Figure S5A) led to a dramatic morphological change (Figures S5B & C) and a reduction in cell number (Figure S5D). Moreover, exogenous SOX2 elevation caused a significant change in the cell cycle distribution of i- SOX2-U87 cells. These cells exhibited an increase in the G2/M PLOS ONE | www.plosone.org 6 August 2012 | Volume 7 | Issue 8 | e44087