1. 10 5 10 6 10 4 10 7 Days of culture N° of viable cells 10 5 10 6 10 4 10 7 10 5 10 6 10 4 10 7 10 5 10 6 10 4 10 7 Supplemental Figure 1. Parental, hTERT immortalized, SV40LT transformed (EHLT) and tansformed tumorigenic EHLT/Ras BJ (human primary foreskin fibroblasts), were treated with 0.5  M of the indicated G4 ligands. At the indicated time points cells were counted and the viability determined. Curves report the number of viable cells in each sample. The mean of three independent experiments with comparable results is shown. Error bars indicate SD. Figure S1

2. Figure S2 BJ-hTERTBJ-EHLT 1  M untreated Supplemental Figure 2: A: BJ-hTERT and BJ-EHLT fibroblasts were exposed to the indicated concentrations of RHPS4 for 24 hours, then fixed in formaldheyde and counterstained with DAPI. Finally, autofluorescence emission of RHPS4 in the cells was analysed at the deconvolution microscope and representative images at 63x magnification are shown. B: Cytofluorimetric analysis of RHPS4 fluorescence intensity in untreated BJ-hTERT and BJ-EHLT (grey area) or in treated BJ-hTERT (orange thick line) and BJ- EHLT (orange dotted line) as above described. A RHPS4 Incorporation Cell Number B

3. Figure S3 50 40 30 20 10 0 381624 _ RHPS4 (hrs) D a b % of  H2AX + cells 10 5 10 6 10 4 10 7 A DNA content BrdU 10%FCS 16,8 % 26,5 % 30,4 % Neg. B Days of culture 10 5 10 6 10 4 % of TIFs + cells N° of viable cells BJ-hTERTBJ-EHLT BJ-hTERT BJ-EHLT RHPS4 Untreated BJ-hTERT BJ-EHLT Supplemental Figure 3. (A) hTERT immortalized and SV40LT transformed (EHLT) BJ fibroblasts, were grown with the indicated concentrations of fetal calf serum (FCS). Progression of cells through the S-phase of cell cycle was analyzed by flow cytometry using BrdU. After 3 days of cell culture, cells were pulsed with 10  M BrdU for 40 min, and the DNA was denatured, incubated with anti-BrdU antibody and the BrdU-positive cells were revealed with FITC-conjugated antibody. The percentage of BrdU-positive cells was reported inside the histogram. (B) In vitro growth curves of untreated or 0.5  M RHPS4-exposed BJ-hTERT and BJ-EHLT cells maintained in medium supplemented with the indicated concentrations of FCS. (C) Cells grown in the indicated concentrations of FCS were treated with 0.5  M RHPS4 for 24 hours starting from the indicated time points from seeding. At the end of treatment each sample was co-immunostained with anti TRF1 and  H2AX antibodies and analyzed. Histograms report the percentage of cells displaying more than 4 TRF1/  H2AX colocalizations (TIFs positive). (D) PHA plus IL-2 activated and proliferating human peripheral blood lymphocytes were exposed to 0.5  M RHPS4. Phase contrast of untreated (a) and RHPS4-treated cells (b) at day 5 of treatment. (E) PHA plus IL-2 activated and proliferating human peripheral blood lymphocytes were treated with RHPS4 for the indicated times (hrs), fixed and processed for IF. The histogram represents the percentage of  -H2AX-positive cells. The mean of three independent experiments with comparable results are shown. Error bars indicate SD. C E 31,2 % 47,8 % Neg. 10%FCS 20%FCS 30%FCS 5%FCS 10%FCS 10 7

4. Figure S4 Untreated RHPS4 4 6 810 0Asynchronous hrs post Thymidine release A 2 %  H2AX+ cells hrs post Thymidine release Hoechst  H2AX merge RHPS4 Untreated CPT 2 hrs post Thymidine release C B Supplemental Figure 4. BJ-hTERT fibroblasts were synchronized at the G1/S boundary with double Thymidine block and released in 0.5  M RHPS4 or in drug-free medium and harvested every 2 hours. Cells were fixed at the indicated times and processed for FACS analysis (A) and for IF against  H2AX (B) Representative images at 63X magnification are reported. Camptothecin treatment (2  M for 2 hours, Sigma) was used as a positive control of  H2AX staining. DAPI was used to mark nuclei. (C) Histograms report the percentage of  H2AX-positive cells. Three independent experiments were evaluated and error bars indicate the SD.

5. Native (G-overhang) Denaturated (Total TTAGGG) A EtBr staining Relative G-signal intensity B E Frequency TFUs 50-100100-150150-200200-250250-300300-350350-400400-450450-500500-550550-600600-650650-700700-750 Figure S5 BJ-hTERT BJ-EHLT/ Ras BJBJ-EHLT BJ-EHLT/Ras BJ-hTERT BJ-EHLT C TRF1 TRF2  -actin Input TRF1TRF2  -actin Alu probe Telo probe D BJ-hTERTBJ-EHLT/ RasBJBJ-EHLTBJ-hTERTBJ-EHLT/ RasBJBJ-EHLTBJ-hTERTBJ-EHLT/ RasBJBJ-EHLT BJ-hTERT BJ-EHLT/ RasBJBJ-EHLT BJ-hTERT BJ-EHLT/ Ras BJ BJ-EHLT BJ-hTERT BJ-EHLT/ Ras BJ BJ-EHLT 25 8 3 1 BJ-hTERT + Exo I Kbp

6. Supplemental Figure 5. (A) Genomic DNA from the various BJ cell lines was digested with HinfI and fractionated on 0.7% agarose gel and then stained with EtBr (right panel). Southern blot analysis for 3′ overhang detection under native conditions (left panel) and telomere detection under denaturing conditions (central panel), by hybridizing gel with (CCCTAA) 4 probe. Where indicated, the DNA was incubated with Exonuclease I (Exo I). Size markers were indicated at right side of EtBr gel. (B) Quantification of the ratio between native and denatured signals from telomeric Southern blot analysis of various BJ cells. Western blot (C) and ChIP analysis (D) of the telomeric proteins TRF1 and TRF2 in BJ cell lines. The levels of  - actin were provided as loading control in western analysis and as negative control in the chromatin IP. The total DNA (input) represents 10% and 1% of genomic DNA. Southern blot analysis was performed by using telomeric (Telo) or ALU repeat-specific probes. A representative experiment is shown on the left panel. The signals obtained were quantified by densitometry, and the percentage of TRF1 (dark gray bars) and TRF2 (white gray bars) precipitated telomeric DNA was calculated as a ratio of input signals and plotted in the graph (right panel). (E) Q-FISH analysis: growing cells were blocked with demecolcine solution to enrich for metaphase cells. Metaphase cells were collected and spread onto glass slices and then processed for Fluorescence In Situ Hybridization with a fluorescent probe against telomeric sequences as described in materials and methods. Fluorescence signals were quantified by the TFL telo software. Histograms report the distribution of the freqence of signal intensities (expressed as TFUs -Telomere Fluorescence Units-) in each population. Three independent experiments were evaluated and error bars indicate the SDs.

7. Figure S6 Untreated RHPS4 Supplemental Figure 6: BJ-EHLT fibroblasts were exposed to 0.5  M RHPS4 for 24 hrs or left untreated and co- immunofluorescence against  H2AX (green) and TRF1 (red) to mark telomeres was performed. Fluorescence signals were analyzed by confocal vertical (x-z) sections (interval: 0.48  m; stack size: 4.3  m) obtained with a Zeiss Confocal Laser Scanning Microscope. (A) A gallery of a representative nucleus for the indicated samples is shown. (B) Average number of  H2AX spots per nucleus co-localizing (telomeric) or not co-localizing (non telomeric) with TRF1. The mean of three independent experiments with comparable results is shown. Error bars indicate  SD. Average number of  H2AX spots per nucleus - RHPS4 + A B Non-telomeric Telomeric

8. Late III Mid EarlyLate I RHPS4 Untreated A Late III Mid EarlyLate I B % of TIFs positive cells (  H2AX/TRF1) C Untreated RHPS4 Supplememtal Figure 7. (A) Cen3 tel human fibroblasts immortalized with hTERT (at early in vitro passages) and their derived transformed phenotypes (from mid to late phase III passages) were treated with 0.5  M RHPS4 for 24 hours and processed for co-immunofluorescence (IF). Percentage of cells containing more than 4 53BP1/TRF1 co-localization foci (considered as TIFs positive) in untreated and treated samples is showed. (B) Representative images at confocal microscope of colocalizations between TRF1 (green) and 53BP1 (red) (Zeiss Laser scan, 60X magnification). Enlarged views of TIF are reported on the bottom of the merged images from Cen3 tel cells of late passages. (C) The indicated cell lines were treated with 0.5  M RHPS4 for 24 hours, processed for co-immunofluorescece against  H2AX and TRF1 and scored for the presence of co-localizing foci. Histograms represent the percentage of cell exhibiting more than 4  H2AX/TRF1 co-localizing foci (TIFs positive) in untreated or treated samples. (D) The indicated cell lines were treated with 0.5  M RHPS4 as above described and growth inhibition was determined after 96 hours of drug exposure. % of phospho-H2AX and TIFs positive cells was assessed in untreated populations as above described. The mean of three independent experiments with comparable results is shown. Error bars indicate SD. Figure S7 % of TIFs positive cells ( 53BP1/TRF1) D