Fig. 4. Effect of FTY720 on brain tumor stem cell (BTSC) invasiveness

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Fig. 4. Effect of FTY720 on brain tumor stem cell (BTSC) invasiveness Fig. 4. Effect of FTY720 on brain tumor stem cell (BTSC) invasiveness. (A) BTSC9 tumor spheres were embedded in a collagen matrix in the presence of 1 μg/mL FTY720 or vehicle (control). Cells invading into the collagen were photographed at the indicated times. (B) After 4 days in the presence of vehicle (control) or of the indicated concentration of FTY720, the area of invasion into collagen was quantitated using the National Institutes of Health ImageJ software. Data are means ± standard deviations from triplicate wells. *Statistically significant difference by Student's t test (P< .01). From: Induction of brain tumor stem cell apoptosis by FTY720: a potential therapeutic agent for glioblastoma Neuro Oncol. 2012;14(4):405-415. doi:10.1093/neuonc/nos005 Neuro Oncol | © The Author(s) 2012. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

Fig. 5. Combination of FTY720 with TMZ Fig. 5. Combination of FTY720 with TMZ. (A) MGMT promoter methylation was examined using methylation-specific polymerase chain reaction (U = unmethylated, M = methylated). (B) BTSC9 or BTSC57 were treated with 0.03 μg/mL of FTY720 or 10 μM of TMZ separately or in combination, and after 4 days, viability was measured by MTT assay. Data are means ± standard deviations from triplicate samples. *Statistically significant difference from FTY720 alone. #Statistically significant difference from TMZ alone by Student's t test. (C) BTSC9 were treated with FTY720 (F) or TMZ (T) at the concentrations listed above separately or in combinations, and lysates were immunoblotted for full-length caspase 7 or GAPDH. From: Induction of brain tumor stem cell apoptosis by FTY720: a potential therapeutic agent for glioblastoma Neuro Oncol. 2012;14(4):405-415. doi:10.1093/neuonc/nos005 Neuro Oncol | © The Author(s) 2012. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

Fig. 6. Combined FTY720 with TMZ in vivo Fig. 6. Combined FTY720 with TMZ in vivo. (A) Representative MRI of tumors in mice with BTSC9 intracranial xenografts 11 days following injection of cells. Mice were treated with vehicle (control), 10 mg/kg FTY720, 5 mg/kg TMZ, or both. (B) Tumor volume was measured as described in Materials and Methods. Data are means ± standard deviations from 3 mice per group. (C) Survival analysis of mice treated as in panel A (5 mice per group). (D) H & E sections showing lack of invasion and well circumscribed tumors in mice treated with FTY720 plus TMZ, compared with control mice or mice treated with either drug alone. From: Induction of brain tumor stem cell apoptosis by FTY720: a potential therapeutic agent for glioblastoma Neuro Oncol. 2012;14(4):405-415. doi:10.1093/neuonc/nos005 Neuro Oncol | © The Author(s) 2012. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

Fig. 1. Partial characterization of brain tumor stem cells (BTSCs) Fig. 1. Partial characterization of brain tumor stem cells (BTSCs). (A) Lysates of the indicated BTSCs were immunoblotted with antibodies specific for CD133. (B) BTSC57 cells were cultured in defined neurosphere medium (NS) or in medium containing 10% FBS. Lysates were blotted for differentiative markers. Longer exposures revealed very low-level GFAP expression in cells grown in neurosphere medium. From: Induction of brain tumor stem cell apoptosis by FTY720: a potential therapeutic agent for glioblastoma Neuro Oncol. 2012;14(4):405-415. doi:10.1093/neuonc/nos005 Neuro Oncol | © The Author(s) 2012. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

Fig. 2. Induction of apoptosis in brain tumor stem cells (BTSCs) by FTY720. (A) Three different BTSC lines were treated with FTY720 for 4 h at the indicated concentrations. Proliferation was measured using either an MTT assay or WST-1 assay according to instructions. Data are means ± standard deviations of triplicate samples. All FTY720 treatments were statistically significantly different from control by Student's t test (P< .05). Three independent experiments provided similar results. (B) Rat primary astrocytes were treated with the indicated concentrations of FTY720, and viable cells were measured by WST-1 assay. (C) Expression of S1P receptors was examined in BTSC9 and BTSC57 cells using quantitative real-time polymerase chain reaction analysis. From: Induction of brain tumor stem cell apoptosis by FTY720: a potential therapeutic agent for glioblastoma Neuro Oncol. 2012;14(4):405-415. doi:10.1093/neuonc/nos005 Neuro Oncol | © The Author(s) 2012. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

Fig. 3. Effect of FTY720 on pro- and anti-apoptotic proteins in brain tumor stem cells (BTSCs). BTSC9 (A), BTSC57 (B), or BTSC44 (C) cells were treated with vehicle alone for 3 h (con) or 4 μg/mL FTY720 for the indicated time, and cell lysates were immunoblotted for regulators of apoptosis. Two independent experiments provided similar results. From: Induction of brain tumor stem cell apoptosis by FTY720: a potential therapeutic agent for glioblastoma Neuro Oncol. 2012;14(4):405-415. doi:10.1093/neuonc/nos005 Neuro Oncol | © The Author(s) 2012. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com