(a) Flow cytometry analysis for the indicated markers in different transformed and wild-type iNPC groups (n=4/group with technical duplicates). (b) Transformation of human iNPCs leads to increased self-renewal properties only in cells where PI3K and MAPK signalling is dysregulated by overexpression of Ras/EGFR/Src mutant genes as highlighted by single-cell self-renewal assays (n=3/group with 24 technical replicates). (c) iNPC transformation results in the upregulation of endogenous NANOG as highlighted by western blot analyses. (d) NANOG expression is upregulated in primary human adult glioma samples. (e) Unsupervised cluster analysis of mRNA expression data highlighting the similarities between transformed iNPCs and primary GTICs. Please note that p53KDiNPCs are more similar to WTiNPCs than to primary GTICs. (f,g) Transformation of human iNPCs induces glycolytic (f) and oxidative phosphorylation changes as indicated by Seahorse analysis (g), as measured by ECAR and OCR (n=4/group with four technical replicates). Data are represented as mean ±s.d. P values were calculated by Student’s t-test or Mann–Whitney test when appropriate and represented as follows: *P<0.05. Scale bars, 200 μm (d).