Fig 2: Model of telomere damage-induced VSMC senescence and inflammation.Stress-induced telomere damage or loss of TRF2 function promotes VSMC senescence and results in leakage of nuclear DNA to the cytosol. Cytoplasmic DNA fragments are then detected by the cGAS cytoplasmic DNA sensor pathway and activate phosphorylation of TBK1 and its downstream effectors, which in turn induce expression of SASP cytokines. Secreted SASP components mediate the recruitment of a variety of immune and inflammatory cells.

Fig 3: DNA-PK kinase activity is not required for Apollo recruitment to telomeres. (A) Immunoblots for HA-Apollo (anti-HA), TRF2 and phosphorylated Chk2 in SV40LT-immortalized TRF2F/F Lig4 -/− MEFs after retroviral transduction transduced with an empty vector (EV) or HA-Apollo, 108 h after Hit & Run Cre-mediated deletion of TRF2 and/or 24 h DNA-PKi treatment. (B and C) Representative immunofluorescence-fluorescence in-situ hybridization (IF-FISH) images and quantification of HA-Apollo localization at telomeres in the same MEFs as described in (A) 120 h after Hit & Run Cre transduction or without any treatment. Apollo and telomeres were detected using anti-HA (red) or Alexa488-OO-(TTAGGG)3 probe (green), respectively. Approximately 300 nuclei were analyzed for each condition over n = 3 independent experiments (100 nuclei per experiment). Median bar is indicated. (D) Immunoblots for HA-Apollo (anti-DCLRE1B, Atlas) and actin in SV40-LT-immortalized DNA-PKcs+/+, DNA-PKcs−/− or DNA-PKcsKD/KD MEFs transduced with an empty vector (EV) or HA-Apollo. * aspecific band. (E and F) IF-FISH analysis and quantification of HA-Apollo localization at telomeres in the SV40-LT-immortalized DNA-PKcs+/+, DNA-PKcs−/− or DNA-PKcsKKD/KD MEFs transduced with an empty vector (EV) or HA-Apollo, analyzed as described in (B, C). For HA-Apollo, 300 nuclei over n = 3 independent experiments are shown (100 nuclei per experiment), with median bar. As negative control, 296 nuclei (at least 90 nuclei per experiment) were analyzed in parallel for EV. (G) Co-immunoprecipitation (Co-IP) of Myc-mTRF2 (WT or F120A) with HA-mApollo in 293T cells without or after 24 h treatment with DNA-PKi. Pull down was performed with anti-HA antibody. HA IP (first and second panels) and Input (third and fourth panels) were analyzed with anti Myc (first and third panels) or HA (second and fourth panels) antibodies. MYC-mTRF2-F120A (F120A) was used as negative control. Statistical analysis by non-parametric Kruskal–Wallis ANOVA test for multiple comparisons (C, F). Statistical significance was indicated by ****P< 0.0001, ***P< 0.001, **P< 0.01, *P< 0.05; n.s., not significant.

Fig 4: EPAS1 promotes TRF1, TRF2, and RAD50 transcription

Fig 5: Proposed model for Apollo recruitment and access to leading-end telomeres after replication. (A) Apollo is recruited at telomeres by TRF2 binding. The physical presence of inactive DNA-PK at the blunt DNA end prevents any nucleolytic attack of the ends, including Apollo’s. After autophosphorylation of DNA-PKcs on the ABCDE/Thr2609 cluster and Apollo interaction with DNA-PKcs, Apollo gains access to the telomere ends and perform the initial resection required for the generation of the 3′overhang and for telomere protection. (B) In the absence of Apollo, when Apollo cannot be recruited by TRF2 or cannot interact with DNA-PKcs, the leading-end telomeres are not resected and are fused via alt-EJ. (C) In the absence of DNA-PK autophosphorylation, due to inhibition of the kinase activity of DNA-PK and/or to mutations affecting the kinase activity and/or the ABCDE/Thr2609 cluster of DNA-PK, Apollo cannot get access to the telomere ends, the leading-end telomeres are not resected and are fused via the alt-EJ.