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Cancer Stem Cells
Version of Record online: 5 JUL 2013
Copyright © 2013 AlphaMed Press
Volume 31, Issue 7, pages 1266–1277, July 2013
How to Cite
Manoranjan, B., Wang, X., Hallett, R. M., Venugopal, C., Mack, S. C., McFarlane, N., Nolte, S. M., Scheinemann, K., Gunnarsson, T., Hassell, J. A., Taylor, M. D., Lee, C., Triscott, J., Foster, C. M., Dunham, C., Hawkins, C., Dunn, S. E. and Singh, S. K. (2013), FoxG1 Interacts with Bmi1 to Regulate Self-Renewal and Tumorigenicity of Medulloblastoma Stem Cells. STEM CELLS, 31: 1266–1277. doi: 10.1002/stem.1401
Author contributions: B.M.: conception and design, collection and/or assembly of data, data analysis and interpretation, manuscript writing, and final approval of manuscript; X.W., R.M.H., and C.V.: conception and design, collection and/or assembly of data, and final approval of manuscript, S.C.M., N.M., S.M.N., C.L., J.T., C.M.F., and C.D.: collection and/or assembly of data and data analysis and interpretation, K.S., J.A.H., M.D.T., and C.H.: data analysis and interpretation; T.G.: provision of study material or patients; S.E.D.: conception and design, provision of study material or patients, collection and/or assembly of data, data analysis and interpretation, and final approval of manuscript, S.K.S.: conception and design, data analysis and interpretation, manuscript writing, and final approval of manuscript.
Disclosure of potential conflicts of interest is found at the end of this article.
first published online in STEM CELLS EXPRESS April 17, 2013.
- Issue online: 5 JUL 2013
- Version of Record online: 5 JUL 2013
- Accepted manuscript online: 17 APR 2013 06:22AM EST
- Manuscript Accepted: 20 FEB 2013
- Manuscript Revised: 27 JAN 2013
- Manuscript Received: 29 SEP 2012
- Canadian Institutes of Health Research Vanier Canada Graduate Scholarship
- Neurosurgical Research and Education Foundation and American Association of Neurological surgeons, Pediatric Section
- Ontario Institute for Cancer Research
- McMaster University Department of Surgery
Additional Supporting Information may be found in the online version of this article.
|sc-12-0914_sm_SupplFigure1.pdf||866K||Supplementary Figure 1: FoxG1 and Bmi1 knockdown lead to an efficient reduction in their transcript and protein levels. shRNA-mediated knockdown of (a) FoxG1 revealed a significant knockdown in shFoxG1-2 construct when compared to cells transduced with a control shRNA construct (n=3, P=0.0047). All other constructs, shFoxG1-1, shFoxG1-3, shFoxG1-4 had varying levels of knockdown but were not significant at transcript level (n=3, P=0.5669, n=3, P=0.2636 and n=3, P=0.1425, respectively). (b) shRNA-mediated knockdown of (b) Bmi1 revealed a significant knockdown in shBmi1-3 construct when compared to cells transduced with a control shRNA construct (n=3, P=0.001). All other constructs, shBmi1-1 and shBmi1-2, had varying levels of knockdown but were not significant at transcript level (n=3, P=0.7923, and n=3, P=0.2064, respectively). (c) Overexpression construct for FoxG1 revealed a significant increase in FoxG1 expression compared to cells transduced with a control construct (n=3, P=0.0018). **P<0.05, ***P<0.005|
|sc-12-0914_sm_SupplFigure2.pdf||1474K||Supplementary Figure 2: Functional characterization of FoxG1 knockdown/overexpression and Bmi1 knockdown constructs through in vitro limiting dilution assays. Characterization of (a) 4 shFoxG1 and (b) 3 shBmi1 constructs with (c) shFoxG1-2 and shBmi1-3 showing the greatest reduction in sphereforming capacity. (d) Cells transduced with OE FoxG1 and OE FoxG1+shBmi1 constructs displayed an increase and decrease in sphere-forming capacity, respectively.|
|sc-12-0914_sm_SupplFigure3.pdf||1231K||Supplementary Figure 3: Representative flow cytometric cell sorting plot for CD15 in Daoy and Med8a medulloblastoma stem cells. (a) Flow plot showing side scatter against forward scatter for Daoy MB stem cells. (b) Flow plot of sort sample showing 5.16% of cells being CD15+. (c) Flow plot showing side scatter against forward scatter for Med8a MB stem cells. (d) Flow plot of sort sample showing 43.29% of cells being CD15+.|
|sc-12-0914_sm_SupplFigure4.pdf||1387K||Supplementary Figure 4: FoxG1 and Bmi1 are differentially enriched in Med8a medulloblastoma cells. (a) FoxG1 (n=3, P=0.00044) and (b) Bmi1 (n=3, P=0.0031) expression are significantly elevated in Med8a MB cells grown in NSC conditions compared to normal human NSCs (n=3). (c) FoxG1 is specifically enriched in CD15+ Med8a MB stem cells compared to CD15- cells (n=5, P=0.00041), however, (d) no difference is observed in Bmi1 expression between CD15+ and CD15- cells (n=5, P=0.7332). (e) Flow plot (left) showing side scatter against forward scatter for Med8a MB stem cells along with (right) FoxG1 and Bmi1 levels showing 99.39% positivity for FoxG1 and 3.12% positivity for Bmi1. **P<0.005, ***P<0.0005|
|sc-12-0914_sm_SupplFigure5.pdf||1005K||Supplementary Figure 5: FoxG1 regulation of MB BTIC differentiation. A general trend demonstrating a (a) decrease in GFAP, MAP2, and OLIG2 expression was observed in OE FoxG1 cells compared to control cells (n=3), whereas an (b) increase in neural differentiation marker expression was observed in shFoxG1 cells when compared to control cells (n=3). (c) Additionally, FoxG1 expression was found to decrease with differentiation in primary human MB BTICs (n=3, P=0.0482). *P<0.05|
|sc-12-0914_sm_SupplFigure6.pdf||2080K||Supplementary Figure 6: Putative MB BTIC marker, CD15, is preferentially expressed in subgroup 3 and 4 MBs. Representative images of differential cytoplasmic CD15 expression in (a) Wnt, (b) Shh, (c) Group 3, and (d) Group 4 MBs. (e) Graphical representation of low/no cytoplasmic CD15 staining to high cytoplasmic positivity within MB subgroups. CD15 staining is marked in non-Shh/Wnt subgroups. (Magnification: ×200)|
|sc-12-0914_sm_SupplTable1.pdf||13K||Supplementary Table 1: Candidate gene list|
|sc-12-0914_sm_SupplTable2.pdf||12K||Supplementary Table 2: Statistical evaluation of candidate genes|
|sc-12-0914_sm_SupplTable3.pdf||7K||Supplementary Table 3: Medulloblastoma stem cell patient isolates: Clinicopathological data|
|sc-12-0914_sm_SupplTable4.pdf||8K||Supplementary Table 4: qRT-PCR & ChIP primers|
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