Auburn, CA) and a-SSEA1-FITC (20 mL/sample, 560127, BD Pharmingen, San Diego, CA). Annexin V was detected with a TACSH Annexin V-FITC Kit (4830-01-K, Trivigen, Inc., Gaithersburg, MD). Neurosphere Growth For neurosphere culture, cells were cultured in bacterial petri dishes in DME/F12 supplemented with 2% B27 (12587010, Invitrogen, Carlsbad, CA), 1% Glutamax (35050061, Invitrogen), 20 ng/mL EGF (E9644, Sigma-Aldrich), 20 ng/mL FGF2 (F0291, Sigma-Aldrich), 20 ng/mL LIF (ESG1107, Millipore, Billerica, MA) and the indicated concentrations of Dox. The cells were observed daily. Results Ectopic SOX2 Expression Impairs Glioblastoma Cell Growth A number of cancers, including glioblastomas, medulloblasto- mas and breast cancers, express SOX2, which has been correlated with poorer prognosis and clinical outcome [4–8]. While Gangemi and colleagues demonstrated that a reduction of SOX2 in glioblastoma tumor cells leads to a significant reduction in cell proliferation [13], it was unclear how glioblastoma tumor cells would respond to rapidly elevated levels of SOX2. To address this question, we initially examined the impact of elevating SOX2 on the physiology of glioblastoma cells. U87 glioblastoma cells were engineered to enable the inducible expression of SOX2 controlled by a tet-responsive element, which could be activated by adding doxycycline (Dox) to the medium. For this purpose, U87 cells were transduced with the FUW- M2rtTA lentiviral vector alone (to introduce a constitutively expressed tet-responsive element) or in combination with the lentiviral vector FUW-tetO-SOX2 (to introduce an ectopic SOX2 transgene under the control of a tet-responsive promoter). Following transduction, cells were refed with culture medium supplemented with increasing concentrations of Dox, and cells were monitored daily. Following 48 hours of SOX2 induction, U87 cell density was dramatically reduced as compared to controls without Dox (Figure 1A). Moreover, elevation of SOX2 led to substantial changes in cell morphology and loss of spindle shaped cells commonly observed in U87 cultures. MTT assays demon- strated that elevated SOX2 significantly reduced cell number at each Dox concentration tested (65% reduction at the highest concentration of Dox tested) (Figure 1B). Importantly, this reduction was not observed in U87 cells transduced with the FUW-M2rtTA vector alone. Remarkably, these dramatic changes in U87 cell physiology occurred following modest increases in SOX2 (2.3-fold) (Figure 1C). To determine whether the dramatic phenotypic changes observed in U87 cells upon ectopic SOX2 expression could be extended to other glioblastoma lines, we performed similar analyses in U118 glioblastoma cells. As in the case of U87 cells, lentiviral vectors were used to deliver FUW-M2rtTA alone or in combination with FUW-tetO-SOX2. Ectopic expression of SOX2 for 48 hours resulted in a dramatic reduction in proliferation and survival of U118 cells, as determined by microscopy observations (Figure S1A). MTT assays demonstrated that elevation of SOX2 led to a significant, dose-dependent reduction in U118 cell number at each Dox concentration tested (Figure S1B). Quantitation of SOX2 levels in U118 glioblastoma cells demonstrated that SOX2 levels rose 2.1-fold one day after addition of Dox to the culture medium (Figure S1C). Together, these findings argue that modest increases (2- to 3-fold) in SOX2 levels substantially disrupt the SOX2 Controls Fate of Brain Tumor Cells in vitro growth of glioblastoma cells and cause dramatic changes in their cell morphology. Thus, SOX2 levels must be carefully maintained within these cells. Elevation of SOX2 in Medulloblastoma Cells Impairs their Ability to Proliferate The effects of elevating SOX2 in glioblastoma led us to examine whether elevating SOX2 also influences the growth of medullo- blastoma tumor cells. For this purpose, DAOY medulloblastoma cells were transduced with the FUW-M2rtTA lentiviral vector alone, or in combination with the FUW-tetO-SOX2 vector described above. Similar to the glioblastoma cells, transduced DAOY cells were cultured in the presence of increasing concentrations of Dox to induce SOX2 expression. Following SOX2 elevation, DAOY cell density was dramatically reduced, as determined by microscopy observations (Figure 2A). Moreover, MTT assays were used to quantify the effects of SOX2, and significant, dose-dependent declines in cell viability were observed (90% reduction at the highest concentration of Dox tested) (Figure 2B). Western blot analysis revealed a ,3-fold increase in total SOX2 levels at 1 mg/mL Dox compared to endogenous levels in the absence of Dox (Figure 2C). The differences observed in the sensitivity of these brain tumor cell lines to exogenous SOX2 (introduced by lentiviral transduction) is likely to be due, at least in part, to differences in the transduction efficiencies of the various cell lines. In this regard, DAOY cells are infected at a higher frequency (,90%) than U118 cells (,63%), as determined by transduction with GFP-expressing lentiviruses and FACS analysis (data not shown). Together, our findings argue that the levels of SOX2 must be carefully controlled to support the growth of U87 and U118 glioblastoma cells, as well as DAOY medulloblastoma cells. Elevating SOX2 Levels Impedes the Growth of both Prostate and Breast Tumor Cells Our findings with glioblastoma and medulloblastoma cells differ from those in other studies where it was reported that elevating SOX2 enhances the growth and tumorigenicity of DU145 prostate tumor cells [19], MCF7 breast tumor cells [18] and A549 lung tumor cells [20]. To determine whether the impairment of cell growth following SOX2 elevation is exclusive to brain tumor cells, we used the same experimental approach described above for brain tumor cells to examine the response of prostate cancer cells to elevation of SOX2. Prostate cancer cells (DU-145) were transduced with lentiviral vectors FUW-M2rtTA and FUW-tetO- SOX2 to introduce a constitutively expressed reverse-tet transac- tivator and a SOX2 transgene under the control of a tet- responsive element, respectively. Following transduction, cells were cultured for 48 hours in medium supplemented with Dox to induce exogenous SOX2 expression (Figure 3C). Interestingly, when SOX2 levels were rapidly induced in DU-145 cells, a marked, dose-dependent reduction in the number of viable cells was observed by both light microscopy (Figure 3A) and MTT assay (Figure 3B). We also examined whether a rapid elevation of SOX2 could perturb the growth of breast cancer cells. For this purpose, MCF7 and MDA-231 cells were transduced with lentiviral vectors FUW- M2rtTA and FUW-tetO-SOX2. Following transduction, cells were cultured for 48 hours in medium supplemented with Dox to induce exogenous SOX2 expression. The increase in SOX2 levels induced in MCF7 (Figure 4) and MDA-231 (Figure S2) cells (,2- to 3-fold) were similar to the levels of SOX2 in MCF7 cells engineered to stably overexpress SOX2 [18], as well as the level of PLOS ONE | www.plosone.org 3 August 2012 | Volume 7 | Issue 8 | e44087