Figure 6. TPP1 mutants fail to rescue TPP1-KO cells from a DNA damage response at chromosome ends.

(A) Immunoblot analysis of the indicated Acd^fl/fl MEF cell lines showing reduction of TPP1 protein levels 72 hours after Cre-ER activation with 4-hydroxytamoxifen (4-OHT) and a uniform rescue of protein levels upon infection with indicated TPP1 WT or mutant lentiviruses. Asterisk indicates nonspecific bands detected by the antibody that serve as loading controls. n = 1. (B) TIF analysis was performed on the cell lines described in A using peptide nucleic acid–FISH for telomeres (red) and immunofluorescence for 53BP1 (green). DAPI was used to stain the nucleus (blue). Appearance of orange foci in the “Merge” panel indicates TIFs. Arrowheads point to 2 representative TIFs in the panel. (C) Quantitation of TIF data of which B is representative. Mean and SD for n = 3 sets of images (each set containing 15–20 cells) are plotted for the indicated cell lines. *P ≤ 0.05 using 2-tailed Student’s t test.