TIMELESS promotes glioma stemness and malignancy through JAK-STAT3 pathway

Timeless was originally identified as a core component of the circadian clock machinery, but its role in GBM stemness and malignancy has not been defined. To investigate the mechanisms underlying Timeless-driven GBM malignancy, we generated CT2A cells with stable Timeless knockdown (KD) using a lentivirus expressing Timeless- specific shRNA, alongside CT2A cells transduced with a non-targeting scramble shRNA (shScramble) as a control.

Total RNA was extracted from three independent biological replicates of shScramble and Timeless-KD CT2A cells, and bulk RNA barcoding and sequencing (BRB-seq) libraries were prepared and sequenced on the Illumina NextSeq2000 platform. Among the genes detected, genes whose mean expression levels in Timeless-KD cells were reduced to half or less of those in shScramble cells were selected and subjected to enrichment analysis using ShinyGO, which revealed significant enrichment of stem cell- related gene signatures.