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Cancer Stem Cells
Version of Record online: 22 MAY 2013
Copyright © 2013 AlphaMed Press
Volume 31, Issue 6, pages 1051–1063, June 2013
How to Cite
Joshi, K., Banasavadi-Siddegowda, Y., Mo, X., Kim, S.-H., Mao, P., Kig, C., Nardini, D., Sobol, R. W., Chow, L. M.L., Kornblum, H. I., Waclaw, R., Beullens, M. and Nakano, I. (2013), MELK-Dependent FOXM1 Phosphorylation is Essential for Proliferation of Glioma Stem Cells. STEM CELLS, 31: 1051–1063. doi: 10.1002/stem.1358
Author contributions: K.J.: collection and/or assembly of data, administrative support, data analysis and interpretation, and manuscript writing; Y.B., S.K., P.M., C.K., D.N., and R.W.S.: collection and/or assembly of data; H.I.K.: provision of study material or patients, interpretation of data, and manuscript writing; L.M.L.C., R.W., and M.B.: collection and/or assembly of data and data analysis and interpretation; I.N.: conception and design, financial support, provision of study, collection and/or assembly of data, 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 February 13, 2013.
- Issue online: 22 MAY 2013
- Version of Record online: 22 MAY 2013
- Accepted manuscript online: 13 FEB 2013 06:19AM EST
- Manuscript Accepted: 29 JAN 2013
- Manuscript Received: 20 AUG 2012
- Ohio State University, Department of Neurological Surgery
- American Cancer Society. Grant Number: MRSG-08-108-01
- National Science Foundation. Grant Number: G.0686.10
- Sontag Foundation Distinguished Scientist Award
- National Institutes of Health (NIH). Grant Numbers: CA148629, GM087798, NS037704, ES019498, GM099213
- NIH. Grant Number: NS0525630
- Sgro/The American Brain Tumor Association
- National Brain Tumor Foundation
Additional Supporting Information may be found in the online version of this article.
|sc-12-0775_sm_SupplFigure1.pdf||315K||Figure S1, related to Figure 1. FoxM1 immunohistochemistry. Immunohistochemistry for FoxM1 (green) and Tuj1 (red) in mouse brains of embryonic day 17.|
|sc-12-0775_sm_SupplFigure2.pdf||101K||Figure S2, related to Figure 1. FoxM1 regulates neural progenitor proliferation. (A)Quantitative RT-PCR analysis of FoxM1b in E17 neurospheres overexpressing FoxM1 (isoforms b) or EGFP (control) (N=3). (B) Left panel representing FoxM1 mRNA expression in E17 neurospheres after transfection with siRNA for either mock, FoxM1a, or FoxM1a/b. The numbers indicate the cycles of PCR reaction. Gapdh is used as internal control. Right panel shows picture representing siRNA treated spheres. Original magnification 10X. (C) Neurosphere forming assay with clonal density of E17 neurospheres overexpressing FoxM1 (isoforms b) or EGFP (control). Neurosphere forming assay were performed in triplicates in 96 well plate and repeated at least three times independently. Scale bars represent 100 micron. (D) Neurosphere forming assay with clonal density of E17 neurospheres after transfection with siRNA for either mock, FoxM1a, or FoxM1a/b. Neurosphere forming assay were performed in triplicates in 96 well plate and repeated at least three times independently.|
|sc-12-0775_sm_SupplFigure3.pdf||223K||Figure S3, related to Figure 2. FOXM1 in mouse GBM model. FoxM1 immunohistochemistry in non-tumor regions of Mut6 mice. Hematoxylin counterstaining is shown in blue. Original magnifications, 20X. Scale bars represent 50 micron.|
|sc-12-0775_sm_SupplFigure4.tif||2669K||Figure S4, related to Figure 2. FOXM1 in human GBM tumors. FOXM1 staining with human GBM tissues, showing nuclear FOXM1 immunoreactivity. Blue staining indicates Hematoxylin counterstaining. Original magnifications, 20X. Scale bars represent 50 micron. Similar staining pattern was observed in 5 different patient samples.|
|sc-12-0775_sm_SupplFigure5.pdf||96K||Figure S5, related to Figure 2. FOXM1 expression in GSCs. (A) Graph (upper panel) indicating the relative FOXM1 mRNA expression determined by qRT-PCR in undifferentiated GBM1600 neurospheres (GSCs) and their sister cultures in pro-differentiation condition (SPGCs). (B) Histograph (lower panel) indicating the relative FOXM1 protein expression determined by flow cytometry with the same sample set described above.|
|sc-12-0775_sm_SupplFigure6.pdf||70K||Figure S6, related to Figure 2: FOXM1 activity in GSCs. Graph indicating the FOXM1 promoter activity in GBM30 GSC vs. SPGCs cells with transfection of the 6×FOXM1 TATA–luciferase plasmid.|
|sc-12-0775_sm_SupplFigure7.pdf||89K||Figure S7, related to Figure 3. Correlation of FOXM1 and MELK expression. Evaluation of correlation of MELK and FOXM1 mRNA expression profile (Affymetrix Human Genome U133A Array) with newly-diagnosed GBM (A) (n=58) and recurrent GBM (B)(n=22).|
|sc-12-0775_sm_SupplFigure8.tif||1346K||Figure S8, related to Figure 3. Transcriptional interaction between FOXM1 and MELK. (A) RT-PCR demonstrating the effect of overexpression of FOXM1, MELK, or GFP (control) on mRNA expression of FOXM1 and MELK. GAPDH is used as internal control. (N=3)|
|sc-12-0775_sm_SupplFigure9.pdf||62K||Figure S9, related to Figure 3. Abundance of GFP protein in MELK immunoprecipitated HEK 293 cells after transfection of the plasmids encoding Empty-Flag, MELK-Flag, and MELK D150A-Flag.|
|sc-12-0775_sm_SupplFigure10.pdf||444K||Figure S10, related to Figure 7. Slice cultures of GBM surgical tissues. Representative H&E staining of a slice culture of GBM surgical tissue treated with DMSO for 72 hours, exhibiting the preservation of the cytoarchitecture of tumors in this assay. Original magnifications, 10X Scale bar represents 100 micron.|
|sc-12-0775_sm_SupplFigure11.pdf||339K||Figure S11, related to Figure 7. Effect of Siomycin A on GBM slice cultures. Effect of Temozolomide (TMZ) or Siomycin A (SM) treatment on cell survival and proliferation using the slice cultures of GBM surgical tissues. Experiment was repeated suing four different patient samples.|
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