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sc-12-0321_sm_SupplFigure1.pdf681KFig.S1. Expression of Survivin is reduced in NS overexpressing Bmi1. Western blot analysis of NestinCre;STOPFloxBmi1 NS culture as compared to control (A) and quantification of the finding by means of densitometry (B). . * p < 0.05 with Student's t test, Error bars represent SD, n=3.
sc-12-0321_sm_SupplFigure2.pdf674KFig.S2. No evidence of Bmi1 binding to the promoter of Survivin in NSCmon. qChIP assay of the Survivin promoter region in NSCmon overexpressing Bmi1 as compared to controls. Data represent means ± SD (n = 3). The antibodies used in qChIP are indicated at the top of each panel; IgG was used as a control. (A,B). The binding activity of each protein is given as percentage of total input.
sc-12-0321_sm_SupplFigure3.pdf2481KFig.S3 Transcriptome analysis of neurospheres overexpressing Bmi1. Complete list of genes differentially expressed in NestinCre;STOPFloxBmi1 NS as compared to controls, Illumina platform mouse v2, n=2 (A). Gene Ontology analysis revealing deregulation of genes involved in differentiation, as well as mitotic cell cycle, selenium binding, cellular and redox homeostasis in these cells. The Table reports the GO Term Category, the number of deregulated genes included in the category (size) and their gene symbols identifier (B). List of a selection of genes differentially expressed in NestinCre;STOPFloxBmi1 NS as compared to controls, SA Bioscience qRT-PCR arrays, n=3 (C).
sc-12-0321_sm_SupplFigure4.pdf1323KFig.S4 Co-localisaion of Survivin and 8OHdG in NSCmon. Immunohistochemical analysis of Survivin (A,D green) and 8OHdG (B,E red) in NSCmon revealed widespread co-localisation of the proteins in both NestinCre;STOPFloxBmi1 cultures and controls (C,F), although the expression of Survivin is stronger in the transgenic cultures. Immunolabelling for γH2AX and Tbr2 on E16.5 neocortex reveals that γH2AX+ cells co-express Tbr2 in keeping with being progenitors committed toward a neuronal lineage (G, K overview and comparative analysis H,I,J and L,M,N). Scale bar is 125μm in A-F and G,M.
sc-12-0321_sm_SupplFigure5.pdf746KFig.S5. Survivin expression levels do not change across NS passages. qRT-PCR analysis of the expression of Survivin across NS passages.
sc-12-0321_sm_SupplFigure6.tif2406KFig.S6. Significant reduction of Bmi1 binding to the promoter of p16Ink4a in mGBM-IC upon Bmi1 knock down. qChIP assay of the p16Ink4a promoter region in mGBM-IC upon Acquati, Greco et al. 2 Bmi1 knock down as compared to si Scr. Data represent means ± SD (n = 3). The antibodies used in qChIP are indicated at the top of each panel; IgG was used as a control. (A,B). The binding activity of each protein is given as percentage of total input. . ** p < 0.01 with Student's t test, Error bars represent SD, n=3. Si Scr: Control, si Bmi1: Bmi1 knock down
sc-12-0321_sm_SupplFigure7.pdf2179KFig.S7 Characterisation of hGBM-IC in xenografts. Histological characteristics of three primary tumour samples showing highly cellular glial tumours (H&E staining A,C, E) with focal vascular proliferations (A, E). The glial origin of the tumours is confirmed by strong and homogeneous positivity of the tumour cells for GFAP, Nestin, Dcx, Olig2 and Sox2 (G,I,K and M,O,Q and S,U,W and Y,A′,C′ and I′,G′,I′ respectively). hBTSC isolated from each of these tumours and injected intracerebrally in NOD-SCID gave rise to tumours with similar histological characteristics (H&E staining, B,D,F). Immunostaining for the same markers mentioned above showed similar expression pattern (H,J,L and N,P,R and T,V,X and Z,B′,D′ and F′,H′,J′respectively). Immunostaining for human vimentin confirmed their origin from the injected cells (K′-P′). Scale bar is 80μm in A-J′ and N′,O′,P′, 2.4mm in K′,L′,M′.
sc-12-0321_sm_SupplFigure8.tif1802KFig.S8. Schematic modelling of epigenetic regulation induced by Bmi1 overexpression at the promoter of target genes. Developmental stage specific differential regulation of p16ink4a and Survivin in neurospheres and NSC is depicted in the upper panel. Similar epigenetic regulation is found in mGBM-IC, lower panel.
sc-12-0321_sm_SupplData.pdf203KSupplementary Data

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