exhibit enhanced growth and tumorigenicity. Importantly, the cells in these reports were studied after selection for their incorporation of drug-resistant transgenes. Thus, these studies did not determine whether the majority of the cells in the tumor population, or only a minor fraction of cells, exhibit increase growth in vitro and in vivo in response to elevated levels of SOX2. In view of the potent ability of SOX2 to dramatically alter the fate of NSC shortly following its elevation, we hypothesized that brain tumor cells must also tightly control the levels of SOX2 in order to support their growth. In this study, we demonstrate that the levels of SOX2 must be maintained below a certain threshold in glioblastoma and medulloblastoma. In this regard, we demonstrate that a 2- to 3-fold increase in SOX2 levels in the population of glioblastoma and medulloblastoma cells decreases their proliferation and alters their cellular morphology. We extended these findings by examining the effects of SOX2 elevation on the physiology of CD133+ and CD1332 subpopula- tions that are present in DAOY cells. We demonstrate that elevating SOX2 impairs the ability of both CD133+ and CD1332 DAOY cells to form neurospheres. We also determined that elevated SOX2 levels causes DAOY cells to exit the cell cycle and express gene markers associated with neural differentiation. Taken together, these studies demonstrate that glioblastoma and medul- loblastoma cells must carefully control the expression of SOX2 to support their proliferation. Finally, we demonstrate similar effects in other types of tumors that express Sox2 endogenously. Two different breast tumor cell lines and a prostate tumor cell line each exhibit decreases in cell proliferation when SOX2 levels are elevated in the cell population from an inducible promoter. The implications of these findings are discussed, especially as they relate to increases in expression of SOX2 during cancer pro- gression. Materials and Methods Cell Culture DAOY medulloblastoma, U87 and U118 glioblastoma, MCF7 and MDA-231 breast cancer, and DU-145 prostate cancer cell lines were cultured in DMEM +10%, as described previously [21]. U87 cells and DU-145 cells were obtained from Webster Cavenee (Ludwig Institute for Cancer Research, La Jolla, CA) and Kaustabh Datta (University of Nebraska Medical Center, Omaha, NE), respectively, who obtained them from the American Type Culture Collection. All other cell lines were obtained directly from the American Type Culture Collection. Plasmid Production Lentiviral packaging vector psPAX2 (Plasmid 12260), envelope vector pMD2.G, (Plasmid 12259), and transfer vectors FUW- M2rtTA (Plasmid 20342) and doxycycline (Dox)-inducible FUW- tetO-SOX2 (Plasmid 20724) were obtained from Addgene (Cam- bridge, MA). G418-selectable pLVX-Tet-OnH Advanced vector was obtained from Clontech (632162, Mountain View, CA). Construction of pLVX-tetO-(fs)SOX2 is described in Methods S1. Lentivirus Production and Cell Transduction Production of lentiviruses and cell transduction has been described previously [22]. After culture in medium containing lentiviruses for 24 hours, the cells were refed with fresh medium. For cells infected with shRNA lentiviruses, cells were selected in medium containing 5 mg/mL puromycin (P8833, Sigma-Aldrich, St. Louis, MO) for 24 hours. SOX2 Controls Fate of Brain Tumor Cells Photomicrographs, MTT Cell Growth Assay and Determination of Cell Number DAOY, U87, U118, MCF7 and MDA-231 cells were sub- cultured into 12-well plates, transduced with appropriate lenti- viruses, and refed with fresh medium containing the indicated concentration of Dox. At the time points indicated, photomicro- graphs were taken with a Canon Rebel XTi camera, and MTT assays were conducted. The protocol for the MTT assay, which uses mitochondrial dehydrogenase activity as a measure of cell number, was described previously [23,24]. Where indicated cell number was also determined with the aid of a Beckman Coulter Counter. Nuclear Protein Isolation and Western Blot Analysis Nuclear proteins were isolated from DAOY, U87, U118, MCF7 and MDA-231 cells using the NE-PERTM kit (Pierce, Rockford, IL), as described previously [25]. Isolated proteins were quantified using a Micro BCA Kit (Pierce), according to the manufacturer’s protocol. Equal amounts of nuclear protein were separated by SDS-PAGE and blotted as described previously [25]. The primary antibodies used were a-SOX2 (ab15830, Abcam, Cambridge, MA) and a-FLAG (F1804, Sigma-Aldrich); the secondary anti- bodies were a-Rabbit IgG-AP conjugate (A3687, Sigma-Aldrich) and a-Mouse IgG-AP conjugate (A4312, Sigma-Aldrich). Relative protein abundance was quantified using ImageQuant 5.2, and small differences in loading were corrected by comparison to the loading control, which was HDAC1 where indicated or a ubiqui- tously expressed non-specific protein band. Generation of i-SOX2-DAOY and i-SOX2-U87 Cells DAOY and U87 cells were transduced for 24 hours with both pLVX-Tet-OnH Advanced and pLVX-tetO-(fs)SOX2 lentiviral vectors. Transduced cells were selected by maintaining cells in culture medium supplemented with 5 mg/mL puromycin for 48 hours. Following puromycin selection, cells were maintained in culture medium supplemented with 300 ng/mL G418 sulfate (631307, Clontech) for 9 days. Cell-Cycle Analysis i-SOX2-DAOY cells were infected as described above, cultured in fresh medium containing Dox for 72 hours, and isolated for cell-cycle analysis. The Telford method was used [26], and FACS analysis was conducted by the UNMC Cell Analysis core facility. RNA Isolation, RT-qPCR Analysis and Microarray Analysis RNA isolation and cDNA synthesis were performed as de- scribed previously [16]. Primer sets for RT-qPCR are listed in Table S1. GeneChip Human Gene 1.0 ST arrays were used for microarray analysis (901085, Affymetrix, Santa Clara, CA). A single Affymetrix chip was used for cells cultured without Dox, and a single chip was used for cells cultured with Dox. Array data has been deposited in the Gene Expression Omnibus repository (Accession No. GSE36947, available at http://www.ncbi.nlm.nih. gov/geo/query/acc.cgi?acc = GSE36947). CD133/SSEA1 Detection and Flow Cytometry Cells were collected, washed twice in stain buffer (0.2% (w/v) bovine serum albumin in PBS), and resuspended at 106 cells/ 100 mL. The cell suspension was supplemented with antibody and incubated for 20 minutes on ice in the dark. Next, the cells were washed twice and resuspended in 1 mL stain buffer. Cells were then FACS sorted/analyzed. The following antibodies were used: a-CD133-APC (10 mL/sample, 130-090-854, Miltenyi Biotech, PLOS ONE | www.plosone.org 2 August 2012 | Volume 7 | Issue 8 | e44087