Gene expression signatures for GSC2 after treated by clofoctol for 6 hours

To address the mechanisms by which clofoctol suppresses growth of GSCs, we performed gene expression profiling using mRNA microarray analysis. GSC2 were incubated with 1‰ DMSO or clofoctol at 3 μM and 10 μM for 6 h before RNA was isolated. Unsupervised hierarchical clustering revealed a high level of consistency across biological replicates. To gain insight into the pathways regulated by clofoctol, we investigated the changes in genes treated with 10 μM clofoctol vs. control using Ingenuity Pathway Analysis (IPA). Diseases and biofunction analysis suggested that clofoctol altered the expression of genes primarily associated with cell apoptosis. Then, to find the crucial genes involved in decreasing cellular growth or inducing cell apoptosis after clofoctol treatment, we analyzed differentially expressed genes. Seventy-six upregulated genes (P <0.05, fold-change >2, FC absolute (10 µM) / FC absolute (3 µM) >3) and 63 downregulated genes (P <0.05, fold-change >2) were differentially expressed in both 3 μM and 10 μM clofoctol treatment groups. To narrow the range of candidates, we then analyzed the differentially expressed genes that showed a relationship with patient survival, according to CGGA (Chinese Glioma Genome Atlas) database, followed by real-time PCR analysis of the expression change in three glioma stem cell lines, GSC2, GSC5, U251 SLC. Only the 5 upregulated genes, LDHB, FGD5-AS1, PDZRN1, KLF13 and NRSN1 were ultimately selected. Gene expression of GSC2 cells was measured at 6 hours after exposure to 0 μM, 3 μM, 10 μM of clofoctol. Three independent experiments were performed at each concentrations.