Chef dr 2 equipment

To prepare agarose plugs we used the protocol reported in [52 (link)] with minor modifications. Briefly, samples were UV irradiated, 6 or 24 h later 1 x 10⁵ cells were melted into 1.0% Pulsed Field Certified Agarose (Bio-Rad Laboratories). Agarose plugs were digested in 0.5 M EDTA-1% N-laurylsarcosyl-proteinase K (1 mg/ml, Invitrogen) at 50°C for 48 h and washed four times in TE buffer and loaded onto a separation gel (1.0% Pulsed Field Certified Agarose). Electrophoresis was performed on CHEF DR II equipment (Bio-Rad Laboratories) as previously described in [52 (link)]. A second electrophoresis protocol was also used [49 (link)], with minor modifications: 9 h, 120°, 5.5 V/cm, 30–18 s switch time; 6 h, 120°, 4.5 V/cm, 18–9 s switch time; 6 h, 120°, 4 V/cm, 9–5 s switch time, for 24 hr. A 2h-bleomycin (100 μg/mL, Gador) treatment was used as a positive control. Ethidium bromide–stained gels were visualized in a White Ultraviolet Transilluminator (UVP) or with Image Quant LAS4000, which allows capture and quantification of images within a linear range. PFGE images were then quantified with the ImageJ software.

Telomere restriction fragments were analyzed using the TeloTAGGG Telomere Length Assay kit (Roche). Briefly, cells were harvested by trypsinization, washed in PBS and collected by centrifugation at 400 g for 4 min. Genomic DNA was isolated using DNeasy Blood and Tissue Kit (Qiagen), digested with HinfI and RsaI restriction enzymes (New England Biolabs) and separated by gel electrophoresis either on 0.8% agarose gels at 50V overnight in 1X TBE buffer or (to resolve elongated telomeres at later time points) on 1% megabase agarose gels (Bio-Rad) using a CHEF DRII equipment (Bio-Rad) under the following conditions: 120° field angle, 5 to 30 s switch times, 5 V/cm and 14°C for 14 hr in 1X TAE. Following the resolution of DNA fragments, DNA was transferred to a positively charged nylon membrane (Roche) by Southern blotting and hybridized with a digoxigenin-labelled telomeric probe. Membranes were exposed to X-ray film (Carestream) and developed in X-OMAT 2000 Processor (Kodak). Mean telomere lengths were calculated as described in Kimura et al. (2010) (link).

Cells were seeded in a 6-well tissue culture plate and treated with either HU (Sigma) for 24 h or SN38 (Abcam) for 6 h/48 h. Media was changed every 24 h during SN38 treatment for 48 h in both treated and untreated cells (DMSO control). Harvested cells were embedded in 2% low melting agarose (Sigma) plugs using CHEF disposable plugs (Bio-Rad). After solidification, plugs were incubated in lysis buffer (100 mM EDTA, 0.2% (w/v) sodium deoxycholate, 1% (w/v) sodium lauroyl sarcosine and 1 mg/ml proteinase K; Promega) at 50°C for 24 h and washed four times in TE buffer (20 mM Tris, pH 8, 50 mM EDTA) for 1 h each. Plugs were run on 1% megabase agarose (Bio-Rad) on a CHEF DR II equipment (Bio-Rad) under conditions of 120 field angle, 5–30 s switch time, 4 V/cm and 14°C for 14 h in 1X TAE. Lambda PFG ladder (48.5–1018 kb; NEB) was used as a molecular size marker. Subsequently, DNA was stained with ethidium bromide and quantification was performed using ImageJ (see figure legends for details).