Ciliary protein ARL13B is a downstream target of EZH2 and its expression correlates negatively with GBM patient prognosis. (A) Schematic diagram of experimental design where GBM43 and GBM6 were treated with either vehicle control or DZNep (0.05 nM) in combination with TMZ (50 µM) for 48 h. Gene expression analysis between the different subtypes of GBM identified ARL13B as one of the top genes for which expression was altered in the absence of EZH2 activity (fold change >6, P < 0.05). (B) Genome-wide ChIP-Seq analysis was performed on GBM43 cells at Days 1 and 4 after TMZ (50 µM) for enrichment of EZH2, H3K27me3 and H3K27ac marks. P-values from Macs2 software called over input control. (C) Quantitative PCR analysis demonstrating that ARL13B expression increases after 4 days of TMZ treatment as compared to vehicle control. Error bars represent mean ± SD of three technical qPCR replicates. Each experiment repeated at least twice. (D) Immunoblot analysis corroborating qPCR data from C at the protein level at 4 days post TMZ treatment. (E) TCGA data demonstrating significant positive correlation between EZH2 expression and ARL13B expression in oligodendroglioma, astrocytoma, and GBM but not in normal brain tissue. (F) TCGA data showing mRNA expression of ARL13B increasing significantly with tumour grade; (G) probability of survival decreasing with higher expression of ARL13B; and (H) time to median survival significantly decreasing with high expression of ARL13B in all brain tumour (top) and only in GBM (bottom). Data were gathered using optimal cut-off. (I) GBM patient tissue samples stained for ARL13B in matched pairs of primary and recurrent tumours demonstrating increased nuclear staining for ARL13B upon recurrence (n = 10). *P < 0.05, **P < 0.01.