Fig 2: 3' overhang and total telomere signal analysis of HEK293T cells expressing POT1 (L259S) and POT1(WT). Southern blots conducted on genomic DNA extracted from HEK293T cells expressing POT1(WT) or POT1(L259S) for 60 d are shown. The gel was probed for TTAGGG signal in both native conditions (to detect single-stranded DNA) and denaturing conditions (to detect total telomeric signal). DNA was also treated with Exo1 to digest 3' ends and detect internal single-stranded DNA (Exo1 resistant). The relative normalized telomeric signal intensity for single-stranded TTAGGG signal is shown below. Source data are available for this figure: SourceData F6.

Fig 3: POT1(L259S) variant patient data. (A) Abridged pedigree of family with POT1 c. 776T>C mutation. Presence of the POT1 c.776T>C; p.(Leu259Ser) gene mutation is indicated by +. (+), presumed carriage of the familial mutation with no DNA available. Circles represent females; squares represent males; symbols with a slash represent deceased individuals. Arrow refers to the proband, roman numerals indicate the generation, and numbers at the left side of the symbols represent the patient numbering in the article. Age at time of diagnosis or age of death is indicated at the upper right of each symbol; TL, telomere length in peripheral blood leukocytes measured by quantitative PCR; TL <1, lower than first percentile of control subjects; TL <10, lower than 10th percentile of control subjects. (B) Patient fibroblast genomic DNA Sanger sequencing shows the heterozygous mutation 776T>C. (C) Telomere length of patients 1–4 measured by a quantitative PCR assay plotted against age. The solid line indicates the 50th percentile of control subjects; the lower dotted lines indicate the first and 10th percentiles of control subjects. (D) HRCT scans of the chest for patient 1 at first visit, classified as probable UIP (pUIP). (E) Patient 2 HRCT at first visit, classified as pUIP pattern. (F) Patient 3 HRCT at first screening visit, classified as pUIP pattern. (G) Patient 4 HRCT at first visit, showing a pUIP pattern.

Fig 4: POT1(L259S) binds DNA with reduced affinity. (A) SDS-PAGE gel of WT and mutant POT1N. (B) FP assays of WT and mutant POT1N. The data show that POT1(L259S) binds telomeric DNA with ~3.5-fold reduced affinity compared with the WT protein. mP, milipolarization. (C) Structure of POT1N (green), DNA (blue/orange; PDB ID: 1XJV). (D) Zoom in at the second OB fold of POT1N and where the mutation L259S is located; same residue and nucleotide numbering as in Lei et al. (2004). Residues (L248, F191) involved in contacts with L259S are shown. Also, K247, which makes a weak hydrogen bond with the backbone of the telomeric DNA, is shown. Source data are available for this figure: SourceData F2.

Fig 5: 53BP1 immunofluorescence reveals increased TIF for POT1(L259S) fibroblasts. (A) Representative images are shown for both control and L259S-containing fibroblasts (scale bar, 5 µm). The green channel represents PNA-FISH probing for telomeric repeats. The red channel shows the immunofluorescence data for 53BP1 antibody staining. The blue color represents DAPI nuclear staining. Colocalizations between telomeres and 53BP1 identified TIFs as shown highlighted by white circles and the adjacent zoomed images. (B) Quantifications are shown for the percentage of cells with at least two TIF signals. Data were obtained from two independent experiments, each performed in duplicate. (C) Total 53BP1 foci were quantified for patient fibroblasts containing POT1(L259S) and control fibroblasts.