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1 DOI: /ncb2774 Figure S1 TRF2 dosage modulates the tumorigenicity of mouse and human tumor cells. (a) Left: immunoblotting with antibodies directed against the Myc tag of the transduced TRF2 forms after the lentiviral infection of BJ-HELT cells. Right: immunoblotting of endogenous TRF2 in cells infected with a lentivirus expressing scramble shrna (shctrl) and an shrna directed against TERF2 (shterf2). Results of one representative experiment are shown. (b) Modulation of TRF2 expression in B16F10 cells. After the lentiviral transduction of B16F10 with lentivirus expressing TRF2 WT, the level of expression of the GFP tag was analyzed by flow cytometry and (c) the level of expression of mterf2 after transduction of B16F10 cells with mtrf2-expressing or mterf2 shrna-expressing lentivirus was determined by RT-qPCR and expressed relative to GAPDH. Results of one representative experiment are shown. (d) Nude mice were injected intravenously with infected BJ-EHLT Ras mc at 1x10 5 cells/mouse. The mean (SE) number of lung nodules on day 50 is indicated. Arrows indicate metastatic nodules. Data from one experiment with n=6 mice per condition are shown and source data are presented in Table S5 (Statistics Source Data). p-values were determined using the Mann-Whitney test. (e) Derivatives of the A375 melanoma cell line injection into nude mice for tumor take. Tumor take was assessed based on palpability; the results are expressed as the percentage of tumor-free mice at the indicated time points after cell injection. n=7 mice per condition, results of one representative experiment are shown and data for two independent experiments are presented in Table S5 (Statistics Source Data). (f) The tumor volume (mean +/- s.d.) was determined and representative images of the tumors are presented in (g). Scale bar represent 1 cm; p-values were determined using the Mann-Whitney test (*p < 0.05; **p < 0.005; ***p < 0.001). 1

2 Figure S2 Partial TRF2 inhibition in BJ-HELTRas cells does not trigger NF-κB, DNA damage and stress response. (a) Anchorage independent growth assay performed 2 weeks pos-infection. Five thousand cells were seeded and the colonies counted three weeks subsequently. Data represent the mean +/- s.d. of n=3 independent experiments and source data are provided in Table S5 (Statistics Source Data). *p<0.05 p-values were determined using the Mann-Whitney test. (b) An increasing number of cells from each sample were plated and colonies were numerated after 10 days. Data represent the mean +/- s.d. of n=3 independent experiments. (c) Growth properties of the indicated cell lines, shown as plots of cumulative population doubling versus the number of days postinfection. Data represent the mean of n=3 independent experiments and source data are provided in Table S5 (Statistics Source Data). (d) NF-κB gel shift analyses using nuclear extracts of the indicated cells. The probe used was a consensus sequence for the transcription factor NF-κB. A nuclear extract of Jurkat cells was used as a positive control. These experiments were performed using a Gelshift NFκB/Rel Family Kit (#37319; Active Motif, Carlsbad, CA). (e) Histology of BJ-HELTRas cells transduced with the indicated lentiviral vector analyzed 7 days after their subcutaneous injection. Biopsies from at least four different mice were analyzed, and at least four sections from each tumor were evaluated. The boxes in the SA-β-galactosidase images represent positive staining in hair follicle and sebaceous gland. See Supplementary Table 2 for quantification. The white bar represent 10 µm. (f) Staining of mouse skin before and after γ-ray irradiation showing the activation of CHK2 and ATM. Immunoblotting of proteins from two TRF2 dn BJ-HELTRas tumors formed in nude mice with anti-chk1 and -phospho-chk1 antibodies showing an absence of detectable DDR activation in contrast to cultured BJ-HELTRas cells treated with the DNA damaging agent campthotecin. The white bar represents 10 µm. All the information about antibodies is reported in Table S4. 2

3 Figure S3 Partial TRF2 inhibition in various tumor cell lines does not uncap telomeres. (a) Chromatin immunoprecipitation experiments performed with BJ-HELT and RasV12 derivatives cell lines five days after infection with the indicated lentiviral vector using γ-h2ax and Myc antibodies and analyzed by slot-blotting hybridized with an Telo or an Alu probe. It is worth noting that we observed a significant binding of the TRF2 dn protein at telomeres, although less pronounced than the one of an equivalent amount of overexpressed myc-trf2. This was unanticipated since TRF2 dn does not contain a DNA binding domain and is believed to work by displacing the endogenous TRF2 molecules. The telomeric binding of TRF2 dn might be explained by an interaction with other shelterin components, data fromone experiment performed in triplicate, all values are plotted on the graph. (b,c) Multiplex fluorescence in situ hybridization analyses (M-FISH) establish that the lines transduced with pwpir-trf2 dn does not show an increased number of chromosome rearrangement, both clonal, i.e. shared by at least two cells, or non-clonal. M-FISH was performed using multi-fish probes (MetaSystems, GmbH) according to the manufacturers recommendations. Images of hybridized metaphases were captured with a CCD camera (Zeisss) coupled to a Zeiss Axioplan microscope and were processed with ISIS software (MetaSystems, GmbH). Data represent the mean +/- S.D. of n=30 metaphases from a single experiment are shown. (d) Telomere length measurement by a Southern procedure. Genomic DNA was digested with HinfI and RsaI and hybridized with a radiolabeled TTAGGG probe, data from 1 experiment are shown. (e) Immunoblotting analysis of shelterin components with the indicated antibodies. (f) Immunoblotting analysis of DNA damage, with the indicated antibodies after exposure to 5 Gy of ionizing radiation. All the information about antibodies is reported in Table S4. (g) Analysis of the phospho-(ser/thr) ATM/ATR substrates. (h) Quantification of the total number of 53BP1 foci per nucleus in the indicated cell lines 5 days post-infection or 5 hours after exposure to 5 Gy of ionizing radiation. Data represents mean+/-s.d. of n=30 nuclei assessed over 2 independent experiments, ***p< p-values were determined using ANOVA test. 3

4 Figure S4 IL-6 is necessary and sufficient to prevent growth inhibition upon TRF2 partial inhibition. (a,b) In the indicated situations, the clonogenic ability is determined as the number of viable colonies issued from the indicated number of plated cells. MOI = Multiplicity of Infection. vil-6 : lentiviral vector expressing full length IL-6; ve : control lentiviral vector not expressing IL-6. Data from 1 experiment performed in triplicate, all values are plotted on the graph. 4

5 Figure S5 The modulation of tumorigenicity by TRF2 dosage correlates with Natural Killer (NK) cell recruitment and activation. (a) Positive controls from the Matrigel assays. PBS alone or containing TNF-α, VEGF-α, SDF-1, and sphingosine-1-phosphate (S1P) was mixed with Matrigel prior to its injection into 5 mice. Five days later, the immune infiltration of the Matrigel plug was assessed using flow cytometry. The min to max box-and-whiskers graph represents the NK cell infiltration of n=5 mice from1 experiment. Data are also presented in Table S5 (Statistics Source Data). p-values were calculated using the Mann-Whitney test (*p < 0.05). (b) Representative FACS dot plots showing CD45 + and CD45 + CD3 + infiltration into the Matrigel plugs quantified in Fig. 5. The data are representative of n=2 independent experiments conducted with 5 mice each. (c) Biopsies from BJ-HELTRas tumor-bearing mice were analyzed by immunohistochemistry. Images showing immunofluorescence staining for NKp46 are shown. The white bar represents 20 µm. (d) Histology of BJ-HELTRas cells transduced with the indicated lentiviral vector and analyzed 7 days after their subcutaneous injection. See Supplementary Table 2 for quantification. All the information about antibodies is reported in Table S4. The white bar represents 10 µm. (e) The expression of TRF2 dn was assessed in tumors formed in NK1.1-treated mice by immunoblotting with anti-myc antibodies, which recognized Myc-tagged TRF2. (f) The percentage of tumor cells expressing TRF2 dn was assayed by tissue analysis with anti-myc antibodies. The values represent the mean percentage of Myc-positive cells from four tumors. Untreated refer to samples of TRF2 dn cells two days after their injection in mice that have not been treated with NK1.1 antibodies. The white bar represents 10 µm. (g) Mice were injected with BJ-HELTRas cells at cells/mouse on day 1. The mice were treated with anti-ifn-γ monoclonal antibodies (clone XMG 1.2) at 0.5 mg/mouse on days 0, 4, and 7. Tumor formation was assessed based on palpability; the results are expressed as the percentage of tumor-free mice at the indicated time points after cell injection following NK cell depletion. n=3 mice per condition. One of two independent experiments is shown. Data for both experiments are presented in Table S5 (Statistics Source Data). (h) Nude mice were injected with i.m. with BJ-HELTRas cells or TRF2 dn overexpressing BJ-HELTRas cells and treated or not with the ATM inhibitor KU After 5 days, microtumors were analyzed by immunohistochemistry for the phosphorylation of ATM, NKp46 infiltration and IFN-g expression. The white bar represents 10 µm. Data for n=2 independent experiments are presented in Table S5 (Statistics Source Data). Quantifications are presented in Figure 6f. 5

6 Figure S6 TRF2 dysfunction in BJ-HELTRas cells does not alter the expression of NKG2D and DNAM-1 ligands and senescence-associated secretory proteins. (a) Analysis of the expression of NKG2D and DNAM-1 ligands on transformed human fibroblasts by flow cytometry. TRF2- or TRF2 dn - BJ-HELTRas cells were incubated at 4 C for 30 min with purified antibodies: anti-mic-a, MIC- B, ULBP-1, ULBP-2, ULBP-3, CD112 and CD155. All the informations about antibodies are reported in Table S4. Representative FACS histograms of 3 independent experiments. (b) The concentration of 24 cytokines was determined in the indicated CM by Luminex Technology using the Milliplex Human Cytokine/Chemokine panel (Millipore Corp., Billerica, MA). The experiment, including sample dilution, beads, antibodies, and cytokine standard preparation, was performed according to the specifications of the manufacturer and run on a Bio-Rad Bio-Plex System (Hercules, CA). Data represent the mean +/- standard deviation of n=3 independent experiments. 6

7 Figure S7 Cells used for assaying HS3ST4 expression did not show DDR activation. Western blot analysis of the phosphorylation of ATM and CHK1 in the samples of BJ-HELTRas cells either overexpressing or down-regulating TRF2 used for the HS3ST4 experiments of Figure 7. All the information about antibodies is reported in Table S4. 7

8 Figure S8 Quantification of CD3+, CD8+ and CD20+ cells in early stages of colon carcinogenesis. Patients assessed for NKp46 and TRF2 staining (Figure 8) where also scored for CD3+ (n=30 low grade adenoma; n=30 high grade adenoma and n=15 focal intramucous adenoma) (a), CD8+ (n=30 low grade adenoma; n=30 high grade adenoma and n=15 focal intramucous adenoma) (b) and CD20+ (n=8 low grade adenoma; n=8 high grade adenoma and n=8 focal intramucous adenoma) (c) infiltration. Data represents mean +/- s.d. of cell densities per mm 2. Data are presented in Table S5 (Statistics Source Data) for the Figure

9 3a 250 kd 100 kd 250 kd 100 kd 72 kd 55 kd 72 kd 55 kd 43 kd 72 kd 55 kd 43 kd 4a 4b Figure S9 Uncropped Western blots. The white boxes refer to the part of the blot presented in the main figure. 9