SupplementaryFigure S1. Expression pattern in GSCs undergoing differentiation. Gene expression levels of markers associated with differentiation of GSCs (neuronal Tuj1, astrocytic GFAP and stem cell-specific CD133) were determined by quantitative RT-PCR. S, GSCs. D, GSCs undergoing differentiation. Histograms represent the mean+S.D. (n=3; *p<0.01, **p<0.05,Student's t test). Data representative of 3 separate experiments.

Supplementary Figure S2. Expression of lineage markers in differentiating GSCs. Undifferentiated GSCs (neurospheres) and GSCs after 4 days of differentiation in the presence of serum were labeled with antibodies against the indicated markers associated with GSCs (Sox2), or with astrocytic (GFAP) or neuronal (Tuj1) differentiation. Scale bar: 10 m.

Supplementary Figure S3. Methylation of CpG sites in the ODZ1 promoter. The methylation status of the indicated three CpG dinucleotides in DNA from two GSC cultures was determined by pyrosequencing. Positions relative to the transcription start site.

SupplementaryFigure S4. ODZ1 modulates cell shape and invasion. (a) F-actin staining of cells differentiated onto a laminin-coated surface. Scale bar: 50 m.(b)Neurospheres containing ODZ1- or empty vector-transfected GSCs were embedded in collagen matrices and the radial invasion of the matrix by tumor cells was visualized by light microscopy. Individual invasion was determined by the mean distance from the edge of the neurosphere to the most distant migrating cells. A representative phase contrast image of a neurosphere is also shown. Histograms represent the mean+S.D. (n=3; *p=0.01, **p=0.007, ***p<0.001, Student's t test). Data representative of 3 separate experiments.

Supplementary Figure S5. ODZ1 expression correlates with lower survival and higher tumor grade. (a) A Kaplan-Meier plot comparing the overall survival of two groups of patients in the Rembrandt data set, with each group having either up- or downregulated expression of ODZ1. (b) Box plot showing the differential expression of ODZ1 in grade III (n=25), grade IV (n=26) and necrotic grade IV (IVnec) (n=50) gliomas based on public dataset (GEO accession code GSE4271). *p=0.024, **p=0.000017, Student's t test. The box represents the values from the lower (25th) to upper (75th) percentile and the middle line indicates the median.

Supplementary Figure S6. Inhibition of RhoA-ROCK blocks ponsin accumulation at focal adhesions. (a-d) The expression of the focal adhesion protein Ponsin is shown in icODZ1-transfected GSCs in the presence or in the absence of different inhibitors of the RhoA-ROCK pathway, Rho inhibitor I, ROCK inhibitor H1152 and siRNAs against ROCK1 and ROCK2. Single plane confocal micrographs were taken under the same intensity conditions. Scale bar: 5 m.

SupplementaryTable S1. Ingenuity Pathway Analysis of genes that modified their levels following expression of ODZ1 (data from the expression microarray), grouped by cellular functions and diseases.

SupplementaryTable S2. Demographic data and clinical characteristics of the 122 patients included in the tissue microarray.

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