Figure 2: Infrequent chromosome end-to-end fusions in HeLa1.2.11 cells overexpressing TRF2.

(a) Representative metaphase spread image of HeLa1.2.11 cells infected with lentivirus expressing GFP or TRF2. Infected cells were passaged and collected at PD7 for metaphase spread followed by FISH analysis. Chromosomes (blue) were hybridized with PNA probes for telomeric sequences (green) or centromeric sequences (red). Regions in white boxes are enlarged to the bottom of the corresponding image for better visualization. Yellow arrows indicate signal-free telomeres; arrowhead indicates chromosome end-to-end fusions. For b and c, 50 metaphases (∼3,360 chromosomes) each of GFP- or TRF2-overexpressing cells were examined for telomeric abnormality. All quantifications were carried out blindly. Each point on the scatter plot represents a single metaphase. Mean values are indicated in red. Two-tailed Student’s t-tests were performed to make pairwise comparison for statistical significance. (b) Quantification of signal-free telomeres in HeLa1.2.11 cells overexpressing GFP or TRF2. (c) Quantification of chromosome end-to-end fusions in HeLa1.2.11 cells overexpressing GFP or TRF2. (d) Schematic diagram of Fusion PCR analysis. (e) Individual chromosome end-to-end fusions assessed by Fusion PCR. HeLa1.2.11 cells overexpressing GFP or TRF2 were harvested at PD6. Multiple aliquots of 100 ng of genomic DNA were independently subjected to fusion PCR using a mix of XpYp, 17p and 21q subtelomeric primers. PCR products were resolved on 1% agarose-TBE gel and detected by Southern hybridization with an XpYp-specific subtelomeric probe. (f) Representative sequence of fusion molecules between XpYp, 17p and 21q. The fusion points, size of deletion, and microhomology (in red) are indicated.