Fig 2: EPAS1 promotes TRF1, TRF2, and RAD50 transcription

Fig 3: 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 4: Stress-induced premature senescence induces persistent telomere damage in human VSMCs.a Experimental timeline of human VSMCs treated with 500 nM doxorubicin for 24 h to induce either acute DNA damage or recovered for 21d to induce SIPS. b Cumulative population doublings (CPD) following doxo 1d + 21d or 1d + 21d controls. c % EdU+ cells following doxo 24 h or 1d + 21d, or 24 h or 1d + 21d controls. d % SAβG or C12FDG-positive cells following doxo 1d + 21d or 1d + 21d controls. e–f qPCR for p16 and p21 and g Western blotting for p16, p21, γ-H2AX, phospho-p53, p53, and 53BP1 in VSMCs following doxo 24 h or 1d + 21d, or 24 h or 1d + 21d controls. h 53BP1 immuno-Telo-FISH DAPI (white), telo-FISH (yellow), and 53BP1 (magenta). Magnified images show colocalizing foci. Scale bar = 5 μm and 1.2 μm (inset). i Mean number of 53BP1 (total) foci or TIFs following doxo 24 h or 1d + 21d or untreated control cells (Unt). More than 30 cells were analyzed/condition per experiment. j Western blot for TRF2 in VSMCs following doxo 24 h or 1d + 21d, or 24 h, or 1d + 21d controls. Data shown are means±SEM, n = 3–5 independent experiments, ns-non significant (p > 0.05), two-tailed Student’s t test or one-way ANOVA, with Dunnett’s correction test.

Fig 5: 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.