SOX2 Controls Fate of Brain Tumor Cells Figure 2. Ectopic elevation of SOX2 in DAOY medulloblastoma cells. (A) Photomicrographs of DAOY 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 DAOY medulloblastoma 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 DAOY 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.g002 Because the proportion of CD133+/SSEA1+ cells increased following elevation of SOX2, we examined the ability of DAOY cells to form neurospheres when SOX2 is elevated. Anchorage- independent growth is often used to assess the tumor-initiating potential of a cell population in vitro [27]. As expected, DAOY cells formed neurospheres in the absence of Dox (Figure S3). In contrast, elevating SOX2 dramatically reduced the capacity of DAOY cells to grow in suspension. Importantly, this observation, along with additional studies presented below, argues that SOX2 impairs the ability of both CD133+ and CD1332 DAOY cells to form neurospheres. Generation of i-SOX2-DAOY Medulloblastoma Cells Although lentiviral transduction of DAOY medulloblastoma was highly efficient (data not shown), the lentiviral vectors used above to characterize the response of brain tumor cells are not well suited to detailed examination of larger numbers of cells, especially when one wishes to examine many different cellular parameters. Consequently, we engineered DAOY cells for the inducible expression of epitope tagged SOX2 using lentiviral vectors containing selection markers, as described in the Materials and Methods. This generated a stably transduced cell population, referred to as i-SOX2-DAOY cells, which express Flag-tagged SOX2 in the presence of Dox (Figure 6A). Importantly, these cells responded to Dox-driven SOX2 induction similarly to our earlier cell populations. Microscopic observations demonstrated that elevation of SOX2 reduced the growth of these cells in a dose- dependent manner, which became clear while following their growth over 4 days. This was confirmed using an MTT assay (Figure 6B). We used i-SOX2-DAOY cells to examine the cell cycle distribution following the induction of SOX2. Elevation of SOX2 in i-SOX2-DAOY cells resulted in significant enrichment of the G2/M (4% to 12%) and G1 compartments (58% to 69%), and a significant decrease in cells in S-phase (38% to 14%) (Figure 6D). Additionally, we observed an increase in the sub-G1 compartment; however, western blot analysis and FACS analysis did not reveal significantly increased levels of cleaved Parp or PLOS ONE | www.plosone.org 5 August 2012 | Volume 7 | Issue 8 | e44087