U2OS (osteosarcoma), HeLa (cervical carcinoma), HT1080 (fibrosarcoma), MCF-7 (mammary adenocarcinoma) and MDA-MB-231 (breast carcinoma) cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco), 1% L-glutamine, 10% FBS (fetal bovine serum); MRC-5 (fetal lung fibroblasts) cells in Minimum Essential Medium Eagle (Sigma), 10% FBS and MCF-10A (mammary epithelial) cells in Mammary Epithelial cell Growth Medium (Lonza) with cholera toxin (at 37 °C with 5% CO2. Cells grown on glass coverslips were fixed in 2% paraformaldehyde/PBS or in methanol: acetic acid (3:1) (Supplementary Fig. S3) and permeabilized with 0.1% triton-X100/PBS. After blocking in 2% Marvel™/PBS, immunofluorescence was performed using standard methods with BG4, anti-FLAG (#2368, Cell Signaling Technology) and anti-rabbit Alexa 594-conjugated (A11037, Invitrogen) antibodies. Coverslips were mounted with Prolong Gold/DAPI (4′,6-diamidino-2-phenylindole) (Invitrogen). For TRF2 detection, anti-TRF2 (ab13579, Abcam) and anti-mouse Alexa 488-conjugated (A11029, Invitrogen) antibodies were used. For enzyme treatments, coverslips were incubated after permeabilization with 120 U of Turbo DNase (0.12 U/μl) or 50 μg/ml of RNase A (Ambion) for 1 h at 37 °C. For pre-incubation experiments, BG4 was incubated with 20-fold excess of pre-folded DNA G-quadruplexes or single-stranded DNA oligonucleotides. Oligonucleotide transfections were performed using 200 nM pre-annealed DNA G-quadruplexes or single-stranded DNA oligonucleotides and TransIT™ Oligo Transfection Reagent (Mirus). For pyridostatin treatment, cells were incubated with 10 μM compound for 24 h. For metaphase preparations, cells were treated with colcemid (50 ng/ml) for 2 h before resuspension in 0.075 mM KCl for 30 min at 37 °C. Cells were then fixed in methanol: acetic acid (3:1) before spreading on slides. 100 well-spread metaphase chromosomes were analyzed for the number and localization of BG4 foci. For cell synchronization, MCF-7 cells were incubated for 24 h in serum-free DMEM (G0/G1), grown for 16 h in DMEM, 20% FBS, 200 μM mimosine (G1/S), and for 3 h in DMEM, 10% FBS (S phase). Cell cycle stages were confirmed using a fluorescent activated cell sorter (FACSCalibur, BD Biosciences). To inhibit DNA replication, cells were incubated with 5 μM aphidicolin for 2 h. Digital images were recorded using a DP70 camera (Olympus) on Axioskop 2 plus microscope (Zeiss), and analyzed with Volocity software (Perkin Elmer). 100-200 nuclei were counted per condition and the standard error mean calculated from 3 replicates. Frequency distribution graphs were plotted using GraphPad Prism (GraphPad Software, Inc.).