(a) Quantification of telomere-bound TRF1 (left panel) and POT1 (right panel) protein levels in immortalized LCLs corresponding to p.R117C carriers and non-carriers. Two independent experiments with replicated samples were performed. a.u.f., arbitrary units of fluorescence. (b) Representative images of TRF1 (red) and POT1 (green) double immunofluorescence in wild-type and p.R117C carriers. White arrows indicate colocalization of both proteins (yellow spots). Scale bar, 5 μm. (c) Quantification of telomeric DNA bound to POT1, TRF1 and TRF2 by ChIP analysis. IgG was used as negative control. Results were normalized to input chromatin. Black bars, wild-type; grey bars, p.R117C carriers. Two independent experiments from each genotype were performed. Lower panel: representative ChIP dot-blot is shown. (d) Left panel: western blot analysis of in vitro-translated FLAG-TPP1, FLAG-TPP1OBD, MYC-POT1, MYC-POT1R117C and MYC-POT1ΔOB1. Right Panel: p.R117C substitution decreased POT1 binding capacity to TPP1. Co-immunoprecipitation assays of the in vitro-translated proteins. FLAG-TPP1 was pulled down with MYC antibody and revealed by FLAG antibody. FLAG-TPP1 lacking the TIN2 and POT1 binding domain, FLAG-TPP1OBD, and a POT1 mutant lacking its OB1 domain were used as controls. Two exposures are showed. (e) p.R117C substitution decreased POT1 binding capacity to telomeric ssDNA. Electrophoretic mobility shift assay of [32P]-labelled oligonucleotide (5′-TTAGGG-3′)7 in the presence of the indicated in vitro-translated POT1 proteins. Data from two independent experiments are shown in d,e. (f) Upper panel: quantification of multitelomeric signal (MTS) events per metaphase in primary lymphocytes by telomeric FISH (n=2 in triplicate). Lower panel: example of MTS (white arrows). Red fluorescence shows telomere signals. Scale bar, 5 μm. (g) Quantification of cells positive for γH2AX (left) and per cent of cells with >5 telomeric induced foci (TIFs) (right) in immortalized LCLs (n=2 in triplicate). Lower panel: representative images of γH2AX and TRF1 immunofluorescence. White arrows show examples of TIFs (yellow spots) with anti-TRF1 (green fluorescence) and anti-γH2AX (red fluorescence). Scale bar, 5 μm. Values are expressed as mean+s.e. The two-tailed student's unpaired t-test was used for the statistical analysis, NS, not significant. DAPI (blue) was used for DNA labelling.