ARL13B regulates cellular plasticity and is required for tumour engraftment in PDX models. (A) Western blot analysis of canonical stem cell markers in U251 GBM cells with wild-type expression, CRISPR-mediated deletion, and lentiviral-mediated rescue of ARL13B. (B) Extreme limiting dilution analysis assay on cells with CRISPR knockdown (KD) of ARL13B or lentiviral rescue of ARL13B compared to non-targeted guide RNA. Error bars demonstrate 95% confidence interval (CI) of stem cell frequency P-value determined from 12-well replicates of plate counting in provided software. (C) Immunoblot analysis showing differential expression of ARL13B, EZH2 an IMPDH2 as well as canonical stem cell markers SOX2 and Oct4 in the neurobasal (10 ng/ml EGF, 10 ng/ml FGF) or differentiation condition media (1% FBS). (D) TCGA data demonstrating mRNA expression of ARL13B across GBM subtypes. (E) Immunoblot analysis showing shRNA mediated knockdown efficiency of ARL13B in GBM5 PDX line. (F–H) In vivo engraftment analysis after removal of ARL13B using shRNA from mesenchymal (F, GBM5), classical (G, GBM6) and proneural (H, GBM43) subtypes of PDX GBM (n = 6 mice per group, 50% male and female mice). *P < 0.05, **P < 0.01, ***P < 0.005.