Fig 1: Wnt signalling pathway is active in PN but not in MES GBM.(a) Gene Set Enrichment Analysis (GSEA) of TCGA data set was performed for the expression of gene components of the canonical Wnt pathway in clinical PN and MES GBMs. Wnt signalling pathway is active in PN GBMs, but not in MES GBMs. (b) Hierarchical cluster analysis on expression data of the Wnt pathway genes differentially expressed between PN and MES spheres. (c) IB analysis of Wnt pathway components including TCF4, FZD6, APC, and activated ß-catenin (ABC) in indicated PN and MES spheres. (d) TopFlash/FopFlash assays that quantify relative Wnt signalling activity in indicated PN and MES spheres. Values in bar graphs were normalized to those in MES 83. (e) Indicated PN and MES spheres were shown expressing GFP under the control of a 7 × TCF/LEF optimal promoter cassette (7 × TOP) and constitutively expressed nuclear mCherry. PN 84 and MES 1123 were treated with Wnt3a (200 ng ml-1), indomethacin (Indo; 20 µM), or a control (vehicle). Scale bar, 100 µm. Bar graph, quantification of fluorescent signal of GFP (TOP-GFP) versus mCherry (PGK-H2BmCherry). The percentage of GFP- and mCherry-positive spheres was determined by FACS. (f) Cell proliferation (upper panels) and sphere formation (lower panels) assays for PN 84 expressing control or specific shRNAs of Wnt3a, Wnt4, and Wnt5a, respectively. Error bars (s.d.) represent the data of triplicate samples for each cell line. *P<0.05, **P<0.01, paired two-way Student's t-test. Data in a–f are representative from three independent experiments with similar results.