Figure 5.

Activation of DNA damage response at telomeres in TERT/TERC mutants. Activation of DNA damage response at telomeres was determined by measuring colocalization of 53BP1 protein and PNA-TelC telomeric probe in VA-13 cells transfected with pBABE empty vector (transfection control), pBABE-WT TERT/TERC (WT telomerase) or with the different TERT and TERC mutants. Potassium bromate (KBrO₃) was used as a positive control. (A) Representative wide-field microscopy images of single cells are shown for those mutants that presented significant differences compared to pBABE-WT TERT/TERC. The red square is magnified in a panel at the right side of the image, to point at telomeres that overlap with 53BP1 (TIF). In blue, counterstaining of nuclei with DAPI. In green, PNA-TelC probe hybridizing with telomere DNA. In red, 53BP1 is a DNA damage marker. (B, C) 53BP1-TelC overlapping index was quantified and normalized to pBABE-WT TERT/TERC for TERT (panel B) and TERC (panel C) mutants. Colors of the bars represent the different gene domains in which the mutations are found: Yellow (controls: pBABE and pBABE-WT TERT/TERC), light green (TEN: p.Leu55Gln to p.Ala202Thr), light blue (RT: p.Val694Met to p.Arg865Cys), orange (CTE: p.Val1090Met and p.Thr1101Met) for TERT and pink (P1a stem: n.23G>C), purple (t/PK: n.96_97delCT and n.98G>A), dark blue (CR4/5: n.269G>A and n.325G>T) for TERC. Graph bars represent mean values ± SEM of two independent experiments. The value of each experiment is the mean 53BP1-TelC overlapping index of five different microscopy fields (average of 200 cells/experiment). Statistical significance between pBABE-WT TERT/TERC and each TERT or TERC mutant was calculated using a two-tailed unpaired t-test (^*P < 0.05; ^**P < 0.01; ^***P < 0.001).