Fig. 3

Distinct telomere maintenance in naïve and primed cells. A Expression levels (CPMs) of telomerase genes Tert and Terc in CBA × C57 naïve and primed cells at P5 and P15. Two biological repeats. B Quantification of telomerase activity of two different naïve and primed cell lines P5 and P15 by ELISA assay. WT N33 ESCs served as telomerase positive controls and telomerase-deficient G4 A49 ESCs served as negative controls. C Relative telomere length shown as T/S ratio by qPCR during passages (P5, P10, and P20) in naïve and primed cells in CBA × C57 or in 129 × C57 background. Four replicates are shown. *P < 0.05; **P < 0.01, ***P < 0.001. D Representative images displaying telomere FISH of naïve and primed cells at CBA × C57 background at P5 and P15. Blue, chromosomes stained with DAPI; green dots, telomeres. The white arrows indicate fragile telomeres. E Histogram shows distribution of relative telomere length displayed as TFU by Q-FISH analysis. Green line indicates medium telomere length. Mean ± s.d. of telomere length is shown above each panel. About 20 chromosome spreads were quantified for each group. Two independent experiments. F Enlarged telomere Q-FISH images, showing telomere fragility (white arrowhead) in primed cells but not in naïve cells. Telomeres were labeled with telomere PNA probes (green), and chromosomes labeled with DAPI (blue). G Frequency of telomere fragility per chromosome. n, number of spreads counted. Two independent experiments. H Expression levels (CPMs) of Trf1, Brca1, and Rtel1 genes in MEF cells, naïve, and primed cells. Two biological repeats. I Representative micrographs showing telomere sister chromatid exchange (T-SCE, red arrows) in naïve and primed cells in CBA × C57 background by CO-FISH analysis. Bottom panel, frequency of T-SCE in the naïve and primed cell lines from three different genetic background. Two independent experiments. Data is shown as Mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001