Extended Data Fig. 4: APC telomeres at T cell chromosome ends.
(a) Representative metaphase spreads showing T cell chromosomes with APC derived telomeres. Scale bar, 0.85 µm. Representative of n = 3 donors. (b) Metaphase spreads generated as in (a) were treated with 1 unit T7 endonuclease for 30 min at 37 °C. The number of T cell chromosomes with APC telomeres before (black values) and after (red values) T7 endonuclease treatment is shown. Pooled from n = 3 experiments (three donors). (c) Presence of APC-derived telomeres in purified nonsenescent T cell plasma membranes from (106) vs T cell nuclei from the same cells 24 h after transfer of fluorescent telomere enriched supernatants derived from APCs activated with ionomycin. The T cell plasma membranes were assessed by confocal imagining on LEICA SP2. APC telomeres were only observed in the nuclear fraction but not in the T cell plasma membrane after the 24 hours incubation. Pooled data from n = 3 independent experiments (three donors). (d) Quantification of APC telomere signal after T cell chromatin immunoprecipitation. Primary human nonsenescent CD3+ T cells (107) were activated by anti-CD3 plus anti-CD28 overnight in in the presence or absence of fluorescent telomere enriched supernatants. The nuclei were digested after lysis with MNase-based Thermofisher Pierce Agarose Chip kit. Chromatin derived from digestion was immunoprecipitated with polyclonal anti-POT1 (1:100) or control rabbit IgG antibodies. The Cy3-fluorescence of APC-derived telomeres was quantified with a microplate reader. Control IP, T cell extracts precipitated with irrelevant IgG. Presence of APC-derived telomeres was confirmed by adding DNase directly to the POT1 IP for 10 min at room temperature prior to fluorescence reading. Pooled results from n = 3 donors. Statistical Tests are provided in the Supplementary Table 1. Error bars indicate S.E.M. throughout.
