Figure 6: Analysis of TUG1 transcripts for maintenance of stemness features of GSCs.

(a–c) Effects of TUG1 overexpression on cell viability (a), apoptosis (b) and expression of the stemness-associated genes (SOX2, MYC, Nestin and CD15) (c) in GSCs treated with γ-secretase inhibitor (RO4929097). Plasmid vectors expressing each TUG1 exon (1–2,132, 2,133–2,910 and 2,911–7,115 nucleotides corresponding to exon 1, exon 2 and exon 3, respectively) were transfected. Viable cells were assessed by trypan blue staining (a). The number of apoptotic cells were counted by FACS analysis with 7-AAD and PE Annexin V staining (b). Expression levels of stemness-associated genes were analysed by qRT-PCR. y-axis indicates relative expression level compared to that seen in DMSO-treated cells (c). Values are indicated relative to abundance in DMSO-treated cells. *P<0.01, Kruskal–Wallis analysis. Empty vector (pcDNA3.1) and plasmid vectors expressing luciferase (Luc) were used for negative controls, whereas NICD overexpression (NICD) was used for a positive control for these experiments. (d,e) Effect of TUG1 overexpression on Nestin activity. Plasmid vectors expressing indicated genes were added to GSC-pE-Nes-222 treated with RO4929097. Phase-contrast and Nestin-EGFP images were shown in (d). Scale bars, 100 μm. (e) Intensity of Nestin-EGFP (left) and number of viable cells (right) compared with the DMSO control were quantified. Viable cells were assessed by trypan blue staining. *P<0.01, Kruskal–Wallis analysis. For all the experimental data, error bars indicate +s.d. (n=3).