SOX2 Controls Fate of Brain Tumor Cells Figure 5. Examination of cell surface markers SSEA1 and CD133 following exogenous expression of SOX2 in DAOY medulloblastoma cells. DAOY cells were infected with FUW-M2rtTA and FUW-tetO-SOX2 lentiviruses and cultured without or with Dox (1 mg/ mL). Twenty-four hours after the addition of Dox, cells were collected and examined for SSEA1 and CD133 surface expression by FACS analysis, as described in the Materials and Methods. doi:10.1371/journal.pone.0044087.g005 no difference in the ability of the i-SOX2-DAOY CD1332 and CD133+ populations to form neurospheres. Moreover, both populations exhibited dramatic reductions in their ability to form neurospheres when Dox was added (Figure 7B), arguing that CD1332 DAOY cells as well as the putative tumor-initiating population of CD133+ DAOY cells must carefully regulate SOX2 levels to support their self-renewal. Our observations suggested that the overall increase in CD133 expression following SOX2 elevation (Figure 5) is due to the upregulation of CD133 surface expression, and not due to the ability of SOX2 to preferentially impair the growth of CD1332 cells. To test this possibility more directly, CD1332 i-SOX2- DAOY cells were isolated and placed in culture in the absence and the presence of Dox. Twenty-four hours after sorting, samples of these cells were subjected to FACS analysis to examine CD133 expression (Figure 8A). As noted above, CD1332 i-SOX2-DAOY cells expressed very low levels of CD133 transcripts. Surprisingly, within only 24 hours, the population grown without Dox had begun to re-express cell surface CD133. Even more remarkable, treatment with Dox caused a large increase in the percentage of CD133+ cells, rising from ,5% to ,60%. To verify that SOX2 was capable of increasing CD133 transcript levels in CD1332 i-SOX2-DAOY cells and to characterize the transcript profile of these cells further, we examined how SOX2 altered the gene expression profile of CD133-low i-SOX2-DAOY cells. For this purpose, FACS sorted CD1332 i-SOX2-DAOY cells were cultured with or without Dox for 24 hours. At that point, RNA was isolated from the two populations. RT-qPCR analysis (Figure 8B) indicated that treatment with Dox caused a large increase in the expression of the SOX2 coding sequence (exogenous plus endogenous) in CD1332 i-SOX2-DAOY cells, but there was little or no difference PLOS ONE | www.plosone.org 8 August 2012 | Volume 7 | Issue 8 | e44087