Fig 2: AKT-dependent TRF1 phosphorylation is required for telomere protection.A. Representative images (above panel) and quantification (below panel) of percentage of ?H2AX positive cells in wild type, TRF1+/T273A, TRF1T273A/T273A, TRF1+/T358A and TRF1T358A/T358A cell lines. Scale bars, 10 µm. B. Representative images (above panel) and quantification (below panel) of percentage of cells presenting three or more ?H2AX and RAP1 co-localizing foci (TIFs) (white arrowhead). Scale bars, 10 µm. C. Representative images (above panel) and quantification (below panel) of multitelomeric signals (MTS) (white arrow heads). Insets represent high magnification images. Scale bars, 5µm. Error bars represent standard deviation. n number of independent experiments (A,B) or number of metaphases (C). Student’s t test was used for statistical analysis, P values are shown. *, p = 0.05; **, p = 0.01; ***, p = 0.001.

Fig 3: AKT-mediated TRF1 phosphorylation regulates TRF1 protein stability.A. Relative TRF1 mRNA expression level in wild-type and in mutant cell lines. B-C. TRF1 wild type and mutant variants (B) and RAP1 (C) turnover was determined in wild type, TRF1T273A/T273A and TRF1T358AT/358A cell lines by cycloheximide (CHX) pulse chase. Cells were treated with 100 µg/ml of CHX in the absence or presence of 5 µM bortezomib. At the indicated times, the cells were harvested to prepare nuclear extracts, followed by Western blot analysis against TRF1 (B) or RAP1 (C) and SMC1 (as loading control) (lower panels). D-E. Phospho-AKT (D) and phospho-S6 (E) levels were determined in wild type, TRF1T273A/T273A and TRF1T358AT/358A cell lines at the indicated times during CHX pulse chase. Total AKT (D) and S6 (E) was used as loading control. The graph represents the mean value for each time point and corresponding standard deviation. n number of independent experiments. Student’s t test was used for statistical analysis, P values are shown. *, p = 0.05; **, p = 0.01; n.s. = not significant.