Extended Data Fig. 1 |. Trf2^−/− ES cells are not viable upon differentiation.

a, Schematic showing the hanging drops protocol used to generate embryoid bodies (EBs) and differentiated FLICs. b, Representative images of embryoid bodies derived from control and TRF2-depleted ES cells before adhesion (left) and 3 days after induction of differentiation (right). The experiment was performed three times with similar results. Scale bar, 400 μm. c, Growth properties of embryoid bodies derived from TRF2-proficient (control (cont.)) or TRF2-depleted ES cells (OHT) undergoing differentiation measured by confluence. Mean and s.d. are derived from the analysis of 47 images per condition. d, Immunofluorescence staining for the pluripotency marker OCT4 in ES cells and FLICs. The percentage of positive cells is shown. The experiment was performed three times with similar results. Scale bar, 10 μm. e, Schematics of the experimental approach used to derive single-cell isolates 3 weeks after OHT-mediated TRF2 deletion in ES cells and FLICs. The table summarizes the number of cells seeded, the number of colonies obtained and the TRF2 genotype of the isolated colonies. An example of a genotyping PCR performed on the resulting clones and the representation of the Trf2-floxed and Trf2-null alleles¹ is shown below. The unprocessed image is provided in the Source Data for Extended Data Fig. 1. f, Growth property measured by confluency of 4 heterozygous ES cells clones (Trf2^f/−) (dark blue) and 4 knockout clones (Trf2^−/−) (red) derived from the experiment described in e, and the parental ES cells (Trf2^f/f) (light blue). Mean and s.d. are derived from the analysis of 36 images per condition.