DOI: 10.1038/ncb3240 Supplementary Figure 1 GBM cell lines display similar levels of p100 to p52 processing but respond differentially to TWEAK-induced TERT expression according to TERT promoter mutation status. (a) GBM cell lines were treated with TNF-α (10ng/ml) for 1h and analyzed for TERT, IL-8 and IκBα expression. Plots depict relative fold change in mrna expression. Data shown represent the mean of 2 independent experiments. (b) Relative Fn14 expression in GBM cell lines carrying either C250T or C228T TERT mutation. Data shown are an average of 2 independent experiments per cell line. Plots depict relative mrna levels. All raw data are shown in Supplementary Table 2. (c) Cytoplasmic and nuclear fractions of untreated or TWEAK-stimulated C250T and C228T GBM cell lines were analyzed for p52 and RelB activation by western blotting with the indicated antibodies. Data shown is representative of two independent experiments. Unprocessed original scans of blots are found in Supplementary figure 7. WWW.NATURE.COM/NATURECELLBIOLOGY 1
Supplementary Figure 2 Canonical NF-κB activation does not induce p65 or p52 recruitment to TERT promoter in GBM cells. (a) ChIP analysis of control (Ctrl) or TWEAK treated GBM cell lines depicting enrichment of BLC promoter with indicated antibodies. n=3 independent ChIP assays performed per cell line. Error bars represent S.D. (b) Western blot analysis of untreated or TWEAK-treated C250T-mutant GBM cells transduced with shrnas targeting p52, RelB, NIK or vector control. Data shown is representative of two independent experiments. (c-d) ChIP was performed in control or TNF-α stimulated GBM cell lines. Enrichment of TERT promoter (c) and NF-κB1A promoter (d) DNA fragments in ChIP DNA were measured by quantitative real-time PCR (ChIP-qPCR) and normalized to DNA input. n=3 independent ChIP experiments performed for each cell line and error bars represent S.D. *P < 0.05; **P < 0.01; Student s t-test, two-tailed. All raw data are shown in Supplementary Table 2. Unprocessed original scans of blots are found in Supplementary figure 7. 2 WWW.NATURE.COM/NATURECELLBIOLOGY
Supplementary Figure 3 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 24h 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 figure 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 anti- LTβR-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. WWW.NATURE.COM/NATURECELLBIOLOGY 3
Supplementary Figure 4 Purified p52 protein binds C250T TERT promoter through its Rel homology domain. (a) Recombinant GST-tagged p52 and GST proteins were analyzed for binding to HIV-κB and C250T TERT promoter DNA-labeled probes with EMSA (right panel). Coomassiestained SDS-PAGE of recombinant p52 protein (left panel). EMSA shown is representative of three independent experiments. (b) EMSA analysis of recombinant wild-type (WT) and mutant p52 (carrying mutation in 2 amino acid residues of Rel-homology domain) proteins binding to HIV-κB and C250T TERT promoter DNA-labeled probes (right panel). Data shown is representative of two independent experiments. Coomassie-stained SDS-PAGE showing amount of WT and mutant p52 proteins used for EMSA analysis (left panel). 4 WWW.NATURE.COM/NATURECELLBIOLOGY
Supplementary Figure 5 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 figure 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 (30ng/ml) 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) (g) and western blot analysis (h). 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 figure 7. WWW.NATURE.COM/NATURECELLBIOLOGY 5
Supplementary Figure 6 Ectopic NIK expression enhances the in vivo tumorigenicity of C250T GBM cells through p52 activation. (a) T98G cells were stably transduced with lentiviral expression constructs for vector, NIK WT and NIK WT in combination with shrna targeting p52 (NIK sh-p52) and total cell lysates were analyzed by western blotting with the indicated antibodies. Representative image from two independent experiments is shown. Unprocessed original scans of blots are found in Supplementary figure 7. (b) 5 NOD-SCID mice were injected subcutaneously with T98G cells expressing vector (red arrows), NIK WT (white arrows) or NIK WT sh-p52 (black arrows) and the tumor growth of these cells in the xenograft mouse model after 10 weeks are depicted. (c) Representative immunohistochemical staining of T98G NIK WT xeonograft tumors with the indicated antibodies. Scale bars, 100μm. (d) Relative telomerase activity of T98G cells expressing vector, NIK WT or NIK sh-p52. Data shown represent n =3 independent experiments here. Error bars represent S.E.M. *P < 0.05; Student s t-test, two-tailed. All raw data are shown in Supplementary Table 2. 6 WWW.NATURE.COM/NATURECELLBIOLOGY
Vector Flag-p52 ETS1/2 Flag IP Flag ETS1/2 Input Flag Actin Figure 6a Supplementary Figure 7 Unprocessed western blot scans. WWW.NATURE.COM/NATURECELLBIOLOGY 7
LN444 LN444 D247mg D247mg Cytoplasmic Nuclear Cytoplasmic Nuclear U251 U251 LN229 LN229 Cytoplasmic Nuclear Cytoplasmic Nuclear p100 p52 80 80 RelA 50 50 RelB GAPDH TRF2 60 50 60 50 Supplementary figure 1c Supplementary Figure 7 continued 8 WWW.NATURE.COM/NATURECELLBIOLOGY
Tweak - + - + - + - + p100 p52 RelB Actin Supplementary figure 2b Supplementary Figure 7 continued WWW.NATURE.COM/NATURECELLBIOLOGY 9
T98G U251 Ctrl Anti-LtβR 24h Ctrl Anti-LtβR 24h p100 p52 Actin Supplementary figure 3a Supplementary Figure 7 continued 10 WWW.NATURE.COM/NATURECELLBIOLOGY
Left panel NIK WT - NIK KK - T98G + - - + Right panel NIK WT NIK KK - - U251 + - - + NIK 160 110 NIK p100 p52 Actin p100 p52 Actin 40 Supplementary figure 5a Supplementary Figure 7 continued WWW.NATURE.COM/NATURECELLBIOLOGY 11
NIK 160 p100 p52 RelB RelA 80 60 Actin Supplementary figure 5h Supplementary Figure 7 continued 12 WWW.NATURE.COM/NATURECELLBIOLOGY
T98G Vector NIK WT NIK sh-p52 NIK 160 110 p100 p52 Actin Supplementary figure 6a Supplementary Figure 7 continued WWW.NATURE.COM/NATURECELLBIOLOGY 13
qpcr primer sequences Gene Name Forward primer (5 - - > 3 ) Forward primer (5 - - > 3 ) Actin CCA TCA CGA TGC CAG TGG TA CCA TCA CGA TGC CAG TGG TA TERT CCA AGT TCC TGC ACT GGC TGA TTC CCG ATG CTG CCT GAC NFkB2 GAC AAG GTG CAG AAA GAT GAC AT TCC GGA ACA CAA TGG CAT ACT RelB AAA CAC ATC AGA GCT GCG GA CCC TGC TGA ACA CCA CTG AT IL8 GCCAACACAGAAATTATTGTAAAGCTT CCTCTGCACCCAGTTTTCCTT IkBα CGCACCTCCACTCCATCC AGCCATGGATAGAGGCTAAGTGTA ETS1 AGACCCTCTCCAGACAGACA GGCGATCACAACTATCGTAGCT ETS2 CCCAGTGGAGCAAGGCAAA AACTCCCATCCGTCTCCAGT Fn14 CCAAGCTCCTCCAACCACAA TGGGGCCTAGTGTCAAGTCT ChIP- qpcr primer sequences Gene Promoter Forward primer (5 - - > 3 ) Reverse primer (5 - - > 3 ) TERT AGGGCCTCCACATCATGGCCCC TCCCAGGGTGCAGGGACGCCAG BLC CTGCGTTGCCTTGACAGTTC GTCGAATGGGCCATCTCTCA TERT +2000 GTCGAGTGGACACGGTGAT AAGTTTATGCAAACTGGACAGGA CRISPR grna sequences grna2- top grna2-bottom grna8-top grna8-bottom 5 -CACCG AGGGGCTGGGAGGGCCCGGA-3 5 -AAACTCC GGGCCCTCCCAGCCCCT C-3 5 - CACCGAAACTCGCGCCGCGAGGAGA-3 5 - AAACTCTCCTCGCGGCGCGAGTTTC-3 Supplementary Table 1 Primer sequences for qpcr and ChIP, and CRISPR grna sequences. Supplementary Table 2 Statistics source data. 14 WWW.NATURE.COM/NATURECELLBIOLOGY