Fig 2: Effects of TLK1 overexpression in U87MG cells. (A) TLK1 mRNA expression following pLOC-TLK1 ORF clone transduction. TLK1 expression was increased by almost 20-fold. Statistical analysis was performed using Kruskal-Wallis test with Dunn's post hoc test. (B) Presence of GFP signals in each cell confirmed successful transduction of pLOC-TLK1 ORF clone was monitored using fluorescence microscopy (magnification, ×100). (C) TLK1 overexpression increased the number of U87MG viable cells by 40%. (D) Overexpression of TLK1 increased proliferative capability of U87MG-pLOC-TLK1 clones. (E) Apoptosis signals were decreased by 50% in TLK1 transduced U87MG cells. (F) Increased BrdU signal in TLK1-overexpressing cells. All experiments were performed in triplicate and the results were compared with the si-NTG control and analysed using a Mann Whitney U test. Values are presented as mean ± standard deviation of 3 biological replicates. *P<0.05, **P<0.01 and ****P<0.001. TLK1, tousled like kinase 1; ORF, open reading frame; RFP, red fluorescent protein; GFP, green fluorescent protein; NTG, non-targeting RNA.

Fig 3: TLK1 knockdown sensitises GBM cells to sublethal dose of TMZ. (A) TLK1 knockdown sensitizes to TMZ at 72 h observed by cell viability assay. (B) Sensitisation effect of TLK1 inhibition in U87MG and LN18 cells colony formation images. (C) Quantitative colony formation assessment of GBM cells treated with si-TLK1 alone and si-TLK1 + TMZ. The colony formation assay confirmed that knockdown of TLK1 sensitised GBM cells to TMZ. Kruskal Wallis was performed with Dunn's multiple correction test. Decreased colony formation observed was due to the decrease in the cellular proliferation and apoptosis. Values are presented as mean ± standard deviation of 3 biological replicates. *P<0.05, **P<0.01, ***P<0.005 and ****P<0.001. TLK1, tousled like kinase 1; GBM, glioblastoma multiforme; TMZ, temozolomide; si-, small interfering RNA; NTG, non-targeting RNA.

Fig 4: TLK1 knockdown in GBM cells decreases cell viability. (A) Reverse transcription-quantitative polymerase chain reaction and western blot analysis were performed to determine transfection efficiency. TLK1 knockdown in both cell lines was effective, with >80% transfection efficiency and decreased TLK1 protein levels at 72 h post-transfection. (B) TLK1 knockdown caused a decrease in the number of viable cells under an inverted light microscope. (C) Significant decreases in viable U87MG and LN18 cells by 40 and 20%, respectively, were observed following TLK1 knockdown at 72 h. (D) Increased apoptotic ssDNA signaling in U87MG cells compared with LN18 cells. (E) Cell cycle analysis indicating a marked increase in S-phase and a decrease in G0/G1 using two-way analysis of variance with Bonferroni's multiple correction post hoc test. All experiments were performed in triplicate and the results were compared with the si-NTG control. Values are presented as mean ± standard deviation of 3 biological replicates. *P<0.05, **P<0.01, ***P<0.005 and ****P<0.001. TLK1, tousled like kinase 1; GBM, glioblastoma multiforme; ssDNA, single-stranded DNA; si-, small interfering RNA: NTG, non-targeting RNA.

Fig 5: Potential hit genes from RNAi screen targeting the GBM kinome in LN18 and U87MG cells. (A) Venn diagram of the 16 and 18 genes identified by the k-Median Absolute Deviation statistical analysis in LN18 cells and U87MG cells, respectively. A total of 4 genes were consistently present in both cell lines: CDC2, TLK1, FLT3 and ALPK1. (B) The complete list of genes identified from RNAi screen. (C) Validation of the 4 genes identified from RNAi screen using Presto Blue assay for cell viability analysis. Readings were performed at 96 h post-transfection. Analysis were performed using Kruskal-Wallis non-parametric test with Dunn's post hoc test. Values are presented as mean ± standard deviation of 3 biological replicates. *P<0.05, **P<0.01 and ***P<0.005. RNAi, RNA interference; GBM, glioblastoma multiforme; TP53, tumor protein 53; CDC2, cyclin dependent kinase 1; TLK1, tousled like kinase 1; FLT3, Fms related tyrosine kinase 3; ALPK1, α kinase 1; si, small interference RNA; NTG, non-targeting RNA.