Figure 7. Overexpression of TRF1-T371D promotes telomere uncapping and results in induction of apoptosis.

(a) Images of metaphase chromosomes depicting telomere abnormalities. Metaphase chromosomes were stained with DAPI and false-colored in red. Telomeric DNA was detected by FISH using a FITC-conjugated (CCCTAA)₃-containing PNA probe in green. Open arrows: telomere doublets. Asterisks represent telomere loss whereas filled arrows indicate sister telomere fusions. (b) Quantification of telomere doublets from TRF1-depleted cells expressing indicated constructs. For each cell line, a total of 3,486 to 4,696 chromosomes from 52 to 71 metaphase cells were scored in a blind manner for the presence of telomere doublets in (b & c) as well as telomere loss in (d) and sister telomere fusions in (e & f). All P values shown in (7b–7f and 7h) were calculated using a Student’s two-tailed t test. Standard deviations derived from three independent experiments are indicated. (c) Quantification of telomere doublets from aphidicolin-treated cells. (d) Quantification of telomere loss from TRF1-depleted cells expressing indicated constructs. Telomere loss refers to chromatids without a detectable telomere signal. The total number of chromatid ends without a detectable telomere signal was divided by the total number of chromatid ends scored, giving rise to the percentage of telomere loss per chromatid end. (e) Quantification of average number of sister telomere fusions per metaphase cell from TRF1-depleted cells expressing indicated constructs. (f) Quantification of percentage of cells with sister telomere fusions from TRF1-depleted cells expressing indicated constructs. (g) Proliferation assays of TRF1-depleted cells expressing various constructs as indicated. Standard deviations from three independent experiments are indicated. (h) Quantification of TUNEL assays. Scoring of TRF1-depleted HeLaII cells expressing various constructs as indicated was done in a blind manner. A total of 1000 cells from each of four independent experiments were scored. Standard deviations are derived from four independent experiments.