SOX2 Controls Fate of Brain Tumor Cells Figure 4. Ectopic elevation of SOX2 in MCF7 breast cancer cells. (A) Photomicrographs of MCF7 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 MCF7 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. ‘*’ indicates statistical significance (p,0.01, student’s t-test). (C) Western blot analysis of SOX2 protein levels in nuclear extracts from MCF7 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.g004 cell cycle compartment (10% to 17%), a reduction in the proportion of cells in the G1 compartment (75% to 64%), and a dramatic increase in cells in the sub-G1 compartment (0.14% to 11%), indicative of cells undergoing apoptosis (Figure S5E). To confirm that i-SOX2-U87 cells were undergoing apoptosis following elevation of SOX2 levels, annexin V staining and FACS analysis was conducted. Similar to our cell cycle analysis, annexin V positive i-SOX2-U87 cells increased in the presence of elevated SOX2 levels from 2.2% to 9.2% (Figure S5F). We then examined whether exogenous SOX2 expression would impair the growth of i-SOX2-U87 cells for a longer period in culture (e.g. 4 weeks), similar to our observations in i-SOX2- DAOY cells (Figure S4). i-SOX2-U87 cells were passaged weekly at clonal density for 4 weeks in total. Similar to our findings in i- SOX2-DAOY cells, elevation of SOX2 in i-SOX2-U87 cells for 1 month impaired their growth dramatically at clonal densities (Figure S6). Thus, both medulloblastoma and glioblastoma cells cannot readily overcome the effects of elevated SOX2 levels, even after 4 weeks in culture. Taken together, although both DAOY and U87 exhibited some proliferative capacity in the presence of exogenous SOX2, i-SOX2-DAOY and i-SOX2-U87 cells were unable to proliferate at the same rate as their uninduced counterparts, even after 4 weeks of culture with elevated SOX2 levels. Exogenous SOX2 Induces CD133 Expression and Gene Markers of Neural Differentiation To more fully characterize the impact of SOX2 on the expression of CD133 in medulloblastoma cells, i-SOX2-DAOY cells (not exposed to Dox) were sorted into CD133-high (CD133+) and CD133-low (CD1332) populations by FACS. RNA was then immediately isolated from both populations. RT-qPCR analysis verified that the CD1332 cell population was virtually devoid of CD133 transcript, especially when compared to the CD133+ population (Figure 7A). Interestingly, levels of SOX2 are slightly higher in the CD133+ population compared to their CD1332 counterparts in DAOY cells, similar to a previous report regarding glioblastoma cells [31]. At the same time, we also cultured the two cell populations on tissue culture plastic (monolayer culture) in medium supplemented with increasing concentrations of Dox. After exposure for 48 hours, both populations exhibited a re- duction in cell number when Dox was added (data not shown). In addition, we examined the two cell populations for their ability to form neurospheres in suspension culture. Remarkably, there was PLOS ONE | www.plosone.org 7 August 2012 | Volume 7 | Issue 8 | e44087