Supplementary Figure 4: Lymphotoxin β receptor (LtβR)-mediated activation of non-canonical NF-κB pathway induces recruitment of NF-κB2 p52 and Pol II to C250T TERT promoter, resulting in enhanced TERT transcription and telomerase function.

(a) Cells were treated with agonistic human LTβR antibody for 24 h and total cell extracts were analyzed by western blotting with indicated antibodies. Data shown is representative of three independent experiments. Unprocessed original scans of blots are found in Supplementary Fig. 7. (b) Relative TERT expression of control (Ctrl) or anti-LTβR-treated T98G and U251 cells. Data from one experiment are shown which is representative of 2 independent experiments. (c,d) ChIP was performed in control (Ctrl) or antiLTβR-treated T98G and U251 cells using p52, p65 or Pol II-specific antibodies and IgG as a negative control. Enrichment of TERT promoter DNA (c) and BLC promoter DNA (d) fragments in ChIP DNA was normalized to DNA input. n = 3 independent ChIP experiments per treatment group and cell line. Error bars represent S.D. (e) Proliferation assay of control (Ctrl) or anti-LTβR-treated T98G and U251 cells. Data shown are from 3 independent experiments for each cell line. Error bars represent S.E.M. (f) Relative telomerase activity of T98G and U251 cells that were untreated (Ctrl) or stimulated with LTβR antibody for 1–4 days. Plots represent mean ± S.E.M. Data shown are from 3 independent experiments for each cell line. (g) Relative TERT expression of T98G and U251 cells treated with si-Control (Ctrl), si-NF-κB2 or si-RelB. Data shown represent the mean of 2 independent experiments. All statistical analyses were performed using Student’s t-test (two-tailed): ^∗P < 0.05; ^∗∗P < 0.01; ^∗∗∗P < 0.001. For raw data, refer to Supplementary Table 2.