Fig 2: AKT-dependent TRF1 phosphorylation is required for proper telomere binding but dispensable for the interaction with other shelterins.A. Representative western blot images (left) and quantification (right) of TRF1, TIN2, POT1, TRF2 and RAP1 protein levels in TRF1+/+, TRF1T358A/T358A and TRF1T273A/T273A cells. B. Representative western blot images (left) and quantification (right) of TRF1, TIN2, POT1, TRF2 and RAP1 protein levels in TRF1+/+, TRF1T358A/T358A and TRF1T273A/T273A cells expressing FLAG-TRF1-WT. C. Total TRF1 levels in wild type HEK293T cells expressing either Flag-TRF1, Flag-TRF1-T273A or Flag-TRF1-T358A by western blot. D. Flag-TRF1, Flag-TRF1-T273A and Flag-TRF1-T358A co-immunoprecipitate with endogenous TIN2 and POT1. Co-immunoprecipitation was performed from lysates of HEK293T cells expressing either the empty vector or the indicated Flag-TRF1 alleles. Quantification of TIN2 and POT1 pulled down with anti-Flag. E-F. Representative images of chromatin immunoprecipitation (ChIP) of telomeric DNA and of ALU sequences with anti-Flag (E) and with anti-TRF2 (F) of HEK293T cells expressing the indicated Flag-TRF1 alleles. DNA input signal is also shown. Quantification of telomeric DNA pulled down with anti-Flag (E) or with anti-TRF2 (F) is shown to the right. ChIP values are normalized by the input of each individual sample. Bars and error bars represent mean values ± SE, n = number of independent experiments. Student’s t-test was used for the statistical analysis; *p < 0.05, **p < 0.01, ***p < 0.001 ns, no significant.

Fig 3: Inhibition of PI3Ka decreases TRF1 levels and increases telomeric DNA damage in human PDXs. a Representative immunoFish images of TRF1 immunofluorescence and telomeric DNA FISH of vehicle and BYL719-treated PDXs. Seven independent tumors were used to generate PDXs in duplicate per condition. Scale bars are shown. b Quantification of telomeric TRF1 foci intensity by immunofluorescence in seven independent PDXs treated either with vehicle or with BYL719 for 12 days. Telomeric TRF1 foci were identified by colocalization with telomeric DNA by FISH. The percent change in telomeric TRF1 intensity in treated PDXs as compared to non-treated are indicated in each case. Seven independent tumors were used to generate PDXs in duplicate per condition. c Representative immunohistochemistry images of phosphorylated AKT (S473) in PDXs treated with either vehicle or with BYL719. Scale bars are shown. d Quantification of pAKT (S473) positive area with regards to total tumoral area in each PDX. Scale bars are shown. The efficacy of the treatment was analyzed by quantifying the percent inhibition of pAKT (S473) as compared to vehicle treated controls and is indicated in each case. e Quantification of mean telomere length by Q-Fish in PDXs treated either with vehicle or with BYL719 for 12 days. f Pearson correlation analysis between telomeric TRF1 foci intensity and pAKT or mean telomere length of untreated and BYL179 treated PDXs. The Pearson r coefficient and the P values are indicated. g, h Number of positive ?H2AX cells (=3 foci) (g) and quantification of telomere-induced foci (TIFs) by double immunofluorescence with anti-RAP1 and anti-?H2AX antibodies (h) in PDXs treated either with vehicle or with BYL719 for 12 days. The PDXs were grouped in non-responders or responders according to the efficacy of BYL179 treatment as measured in d. i Representative RAP1 and ?H2AX immunofluorescence images of vehicle and BYL719-treated PDXs. Damage cells (green) are marked with a white arrow and TIFs (yellow) with a yellow arrow head. Scale bars are shown. Student’s t test was used for statistical analysis and P values are shown. Error bars represent standard errors. n number of independent experiments

Fig 4: Disruption of the interaction between RAP1 and SUN1 does not interfere with APB formation in ALT cells. (A) U2OS cells were infected with the knockdown control (shLuc) or shRAP1 lentivirus and simultaneously complemented with control (EV), wild-type RAP1 (WT), RAP1 coil deletion (?Coil), nonphosphorylatable (8FA), or phospho-mimetic (8DE) RAP1 mutant lentiviruses. Virus-infected cells were selected for 5 days and subjected to further methionine restriction for 3 days. Cell lysates were analyzed by immunoblotting with anti-RAP1 and anti-GAPDH antibodies. The arrowhead indicates the location of endogenous RAP1, and the multiple lower-molecular-weight bands are degraded RAP1. Asterisk (*), RAP1 coil deletion mutant. GAPDH was used as the loading control. (B) Quantification of APBs (%) in the U2OS cells shown in (A). Approximately 200-300 cells were analyzed for each independent experiment. Error bars denote SD; n=3 (independent experiments); *P<0.05 (two-tailed Student’s t-test). N.S., no significance.

Fig 5: RAP1 protects telomeres against DNA damage Western blotting showing the expression of RAP1 and TRF2 in MRC-5 cells of different population doublings (PD). Senescent cells (PD 72) were left in culture for at least 4 weeks before harvesting for analysis.ChIP analysis of young (PD 30) and senescent (PD 72) MRC-5 cells performed with either anti-RAP1, anti-TRF2, or an IgG antibody. The immunoprecipitated and input products were loaded into a slot blot membrane and hybridized with a telomere probe, and stripped and hybridized to an Alu probe in order to determine unspecific binding. Quantification was performed by normalizing the telomere signal of the immunoprecipitated to that of the input. Data represent mean ± SD of three biological replicates.Immunofluorescence detection of 53BP1 (red) and a FISH probe staining telomeres (green) in young (PD 26), pre-senescent (PD 66), and senescent (PD 72 + 4 weeks) MRC-5 fibroblasts. The upper graph shows the percentage of telomere co-localizing with 53BP1 (TIFs). Total DNA damage was measured by immunofluorescence detection of 53BP1. The lower graph shows the total number of 53BP1 foci per nucleus. Approximately 40–50 cells were analyzed per replicate and per condition. Bars represent SEM of two biological replicates.Data information: Statistical analyses were performed using Mann–Whitney U-test (*P < 0.05; **P < 0.001; ***P < 0.0001). Scale bar = 10 µm.Source data are available online for this figure.