Supplementary Figure 6: Constitutive expression of NF-κB-inducing kinase (NIK) results in transcriptional activation of C250T TERT promoter, which promotes the telomerase activity of GBM cells.

(a) T98G and U251 cells were transfected with vector, human NIK wild-type (WT) or kinase-inactive mutant NIK (KK) expression plasmids and total cell lysates were analyzed by western blotting with the indicated antibodies. Data shown is representative of three independent experiments. Original scans of blots are shown in Supplementary Fig. 7. (b) ChIP was performed in control (Ctrl) or NIK WT-overexpressing T98G and U251 cells using p52, p65 or Pol II-specific antibodies and IgG as a negative control. Enrichment of TERT promoter DNA fragments in ChIP DNA was normalized to input. n = 3 independent ChIP experiments per cell type. Error bars represent S.D. (c) Relative telomerase activity of T98G and U251 cells transfected with vector, NIK WT or NIK KK constructs. Data from one experiment is shown which is representative of 3 and 2 independent experiments for T98G and U251 cells respectively. (d) ChIP analysis of control or NIK WT-overexpressing T98G and U251 cells depicting enrichment of BLC promoter with indicated antibodies. n = 3 independent ChIP experiments per cell type and error bars represent S.D. (e) Luciferase reporter assays were performed in 293T HEK cells that were co-transfected with empty vector or human NIK expression plasmid and the pGL3 basic reporter vector or pGL3 vector containing either the WT TERT promoter region (−340 to −55) or TERT promoter with C250T mutation (−340 to −55). Data shown represent the mean luciferase activity from 2 independent experiments. (f) Luciferase reporter assays were performed in 293T HEK cells that were transfected with pGL3 empty reporter vector or pGL3 vector containing either the WT TERT promoter region or TERT promoter with C250T mutation and subsequently untreated or stimulated with TWEAK (30 ng ml⁻¹) for 1D. Luciferase assay data shown are from one of two independent experiments. (g,h) Vector- or NIK WT-expressing T98G cells were transfected with si-Ctrl, si-RelB or si-NF-κB2 and analyzed for relative TERT expression (average fold change ± S.D.;n = 3 independent experiments). Western blot data is representative of two independent experiments. ^∗P < 0.05; ^∗∗∗P < 0.001; Student’s t-test, two-tailed. All raw data are shown in Supplementary Table 2. Unprocessed original scans of blots are found in Supplementary Fig. 7.