in vitro and in vivo in response to elevated SOX2. This does not diminish the importance of SOX2 expression in prostate, breast or brain tumor cells. Rather, it strongly suggests that SOX2 levels in these tumors are unlikely to rise in isolation, but must be accompanied by other changes in gene expression that work in cooperation with SOX2 to enhance the growth of tumor cells. Importantly, we believe future studies should be directed toward the identification of changes in gene expression that work cooperatively with SOX2. Identifying genes required by SOX2 for growth promotion may offer new therapeutic targets for the treatment of SOX2-dependent cancers. Additionally, our findings illustrate the advantages offered by the use of inducible promoters when studying the roles of specific genes in cellular physiology. In conclusion, the findings described in this study underscore the importance of carefully regulating the expression and function of the essential transcription factor SOX2 in brain tumor cells. Understanding how SOX2 levels are carefully controlled in tumor cells will provide novel insights into the fundamental biology of these cancers. Moreover, this understanding may help identify new targets for the development of improved therapeutic approaches to treat these devastating cancers. Supporting Information Figure S1 Ectopic elevation of SOX2 in U118 glioblas- toma cells. Photomicrographs of U118 cells infected with FUW- M2rtTA or FUW-M2rtTA and FUW-tetO-SOX2 lentiviruses, cultured in various concentrations of Dox. Photomicrographs were taken 48 hours after the addition of Dox to the culture medium. (B) MTT assay of U118 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 U118 cells infected with FUW-M2rtTA and FUW-tetO-SOX2 lentiviruses, and cultured without or with Dox for 24 hours to induce SOX2 expression. (TIF) Figure S2 Ectopic elevation of SOX2 in MDA-231 breast cancer cells. (A) Photomicrographs of MDA-231 breast 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 MDA- 231 breast 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. ‘*’ and ‘**’ indicate statistical significance (p,0.01 and p,0.001, respectively, stu- dent’s t-test). (C) Western blot analysis of SOX2 protein levels in nuclear extracts from MDA-231 cells infected with FUW-M2rtTA and FUW-tetO-SOX2 lentiviruses, and cultured without or with Dox for 24 hours to induce SOX2 expression. (TIF) Figure S3 Elevated SOX2 levels impair neurosphere growth of DAOY cells. Photomicrographs of DAOY medullo- blastoma cells infected with FUW-M2rtTA or FUW-M2rtTA and SOX2 Controls Fate of Brain Tumor Cells FUW-tetO-SOX2 lentiviruses, cultured as neurospheres, in various concentrations of Dox. Photomicrographs were taken five days after cultures were set up. (TIF) Figure S4 Long-term growth of i-SOX2-DAOY cells expressing exogenous SOX2. i-SOX2-DAOY cells were grown in the absence and presence of Dox (1 mg/mL) for 4 weeks. Cells were subcultured weekly, at which time the Dox-treated and control populations were seeded at the same density. Photomicro- graphs of Dox-treated and control cells were taken at the end of the 4th week after being plated at the indicated cell densities (cells per T25 culture flask). (TIF) Figure S5 Characterization of i-SOX2-U87 glioblasotma cells. (A) Western blot analysis confirming Dox-inducible expression of flag-epitope tagged SOX2. i-SOX2-U87 cells were exposed to Dox for 24 hours and nuclear extracts were isolated. Photomicrographs of i-SOX2-U87 cells following (C) 72 and (D) 96 hours exposure 6 Dox (1 mg/mL). (D) Determination of cell number was determined by MTT assay and direct cell counts with the aid of a Beckman Coulter Counter. In each case, three control samples and three Dox treated samples were analyzed. ‘*’ and ‘**’ indicate statistically significant difference (p,0.01 and p,0.001, respectively, student’s t-test) between untreated and Dox-treated samples. (E) Cell cycle analysis of i-SOX2-U87 cultured in the absence or presence of Dox (1 mg/mL) for 48 hours. Data for cells without Dox was collected as independent duplicates; whereas, samples with Dox were collected in triplicate. Error bars represent standard deviation. The student’s t-test was used to determine p- values. (F) Annexin V staining of i-SOX2-U87 cells cultured in the absence and presence of Dox for 48 hours. (TIF) Figure S6 Long-term growth of i-SOX2-U87 cells ex- pressing exogenous SOX2. i-SOX2-U87 cells were grown in the absence and presence of Dox (1 mg/mL) for 4 weeks. Cells were subcultured weekly, at which time the Dox-treated and control populations were seeded at the same density. Photomicro- graphs of Dox-treated and control cells were taken at the end of the 4th week after being plated at the indicated cell densities (cells per T25 culture flask). (TIF) Primer sets used for RT-qPCR analysis. Table S1 (XLS) Table S2 Microarray data from DAOY cells in the absence (2Dox) and presence (+Dox) of exogenous SOX2 expression. (XLS) Methods S1 (DOC) Acknowledgments Michel Ouellette is thanked for advice regarding the examination of senescence. Author Contributions Conceived and designed the experiments: JLC PJW AR. Performed the experiments: JLC PJW MD. Analyzed the data: JLC PJW AR. Contributed reagents/materials/analysis tools: JLC PJW MD AR. Wrote the paper: JLC PJW AR. PLOS ONE | www.plosone.org 13 August 2012 | Volume 7 | Issue 8 | e44087