Telomere shortening induces micronuclei formation and transposon mobilization.

(A) Quantification of mean telomere length measured by TRF analysis in the skin (n_WT = 5, n_tert-/- = 5, n_sting-/- = 5, n_{tert-/- sting-/-} = 4, WT vs tert-/-p = 0.017, tert-/- vs sting-/-p = 0.002, sting-/- vs tert-/- sting-/-p = 0.008). (B) Quantification of mean telomere length measured by TRF analysis in the testis (n_WT = 4, n_tert-/- = 6, n_sting-/- = 5, n_{tert-/- sting-/-} = 6, WT vs tert-/- sting-/-p = 0.008). (C) Quantification of mean telomere length measured by TRF analysis in the kidney marrow (n_WT = 3, n_tert-/- = 4, n_sting-/- = 7, n_{tert-/- sting-/-} = 7, WT vs tert-/-p = 0.000003, WT vs tert-/- sting-/-p = 0.0008, tert-/- vs sting-/-p = 0.000001, sting-/- vs tert-/- sting-/-p = 0.0005, tert-/- vs tert-/- sting-/-p = 0.003). (D) Quantification of mean telomere length measured by TRF analysis in the intestine (n_WT = 6, n_tert-/- = 6, n_sting-/- = 6, n_{tert-/- sting-/-} = 6, WT vs tert-/-p = 0.0007, WT vs tert-/- sting-/-p = 0.015, tert-/- vs sting-/-p = 0.0004, sting-/- vs tert-/- sting-/-p = 0.008). In each boxplot (A–D), the boxes represent the interquartile range (IQR; 25th to 75th percentile). Inside each box, the horizontal line indicates the median. The whiskers extend to the most extreme data points (minima and maxima) within 1.5 × IQR from the quartiles. (E) Representative immunofluorescence images and quantifications of MN formation in the fibroblasts derived from skin (n_WT = 1, n_tert-/- = 1, n_sting-/- = 1, n_{tert-/- sting-/-} = 1, WT vs tert-/-p = 0.040, WT vs tert-/- sting-/-p = 0.034, tert-/- vs sting-/-p = 0.047, sting-/- vs tert-/- sting-/-p = 0.040). The yellow arrows point at the micronuclei. Scale bar = 5 µm. (F) Representative western blot images and quantification of H3K9me3 in the skin (n_WT = 6, n_tert-/- = 6, WT vs tert-/-p = 0.032). (G) RT-qPCR analysis of ltr-2 gene expression (n_WT = 3, n_tert-/- = 3, WT vs tert-/-p = 0.038). Data in Fig. 1E–G are presented as the mean ± s.e.m.; *p < 0.05; **p < 0.01, ***p < 0.001, using a one-way ANOVA and post hoc Tukey test (panel A–E), or unpaired t-test (panel F, G). Source data are available online for this figure.

Telomere shortening activates the cGAS-STING pathway.

(A) Representative western blot images and quantification of downstream targets of cGAS-STING pathway (n_WT = 4–5, n_tert-/- = 5–6, n_sting-/- = 5–7, n_{tert-/- sting-/-} = 5, p-Irf3/Irf3: WT vs tert-/- p = 0.049, tert-/- vs tert-/- sting-/- p = 0.011; p-Tbk1/Tbk1: WT vs tert-/- p = 0.035, tert-/- vs sting-/- p = 0.002, tert-/- vs tert-/- sting-/- p = 0.042). (B) RT-qPCR analysis of isg15 gene expression in the skin, testis, kidney marrow and intestine (n_WT = 5–6, n_tert-/- = 4–8, n_sting-/- = 5–8, n_{tert-/- sting-/-} = 7–9, skin: WT vs tert-/- p = 0.024, sting-/- vs tert-/- p = 0.011, N = 5–7, tert-/- vs tert-/- sting-/- p = 0.007; testis: WT vs tert-/- p = 0.001, sting-/- vs tert-/- p = 0.001, tert-/- vs tert-/- sting-/- p = 0.002; kidney marrow: N = 6–9, WT vs tert-/- p = 0.045, sting-/- vs tert-/- p = 0.022, tert-/- vs tert-/- sting-/- p = 0.019; intestine: WT vs tert-/- p = 0.0008, sting-/- vs tert-/- p = 0.0007, tert-/- vs tert-/- sting-/- p = 0.001). (C) RT-qPCR analysis of ifn-i gene expression in the skin, testis, kidney marrow and intestine (n_WT = 4–6, n_tert-/- = 5–8, n_sting-/- = 5–8, n_{tert-/- sting-/-} = 6–9, skin: sting-/- vs tert-/- p = 0.004, tert-/- vs tert-/- sting-/- p = 0.002; testis: WT vs tert-/- p = 0.003, sting-/- vs tert-/- p = 0.003, tert-/- vs tert-/- sting-/- p = 0.003; kidney marrow: WT vs tert-/- p = 0.008, sting-/- vs tert-/- p = 0.004, tert-/- vs tert-/- sting-/- p = 0.011; intestine: WT vs tert-/- p = 0.020, sting-/- vs tert-/- p = 0.005, tert-/- vs tert-/- sting-/- p = 0.014). Data were presented as the mean ± s.e.m.; *p < 0.05; **p < 0.01, ***p < 0.001, using a one-way ANOVA and post hoc Tukey test. Source data are available online for this figure.

cGAS-STING pathway inactivation attenuates DNA damage response.

(A) Representative immunofluorescence images of DNA damage. Scale bar = 10 µm. (B) Quantification of DNA damage in skin (n_WT = 6, n_tert-/- = 6, n_sting-/- = 6, n_{tert-/- sting-/-} = 6; WT vs tert-/- p = 0.00001, sting-/- vs tert-/- p = 0.000001, WT vs tert-/- sting-/- p = 0.000001, sting-/- vs tert-/- sting-/- p = 0.000003). (C) Quantification of DNA damage in testis (n_WT = 6, n_tert-/- = 6, n_sting-/- = 6, n_{tert-/- sting-/-} = 6, N = 5–6, WT vs tert-/- p = 0.000001, sting-/- vs tert-/- p = 0.000001, WT vs tert-/- sting-/- p = 0.000001, sting-/- vs tert-/- sting-/- p = 0.000002). (D) Quantification of DNA damage in kidney marrow (n_WT = 6, n_tert-/- = 6, n_sting-/- = 6, n_{tert-/- sting-/-} = 6; WT vs tert-/- p = 0.00004, sting-/- vs tert-/- p = 0.0001, WT vs tert-/- sting-/- p = 0.0001, sting-/- vs tert-/- sting-/- p = 0.0003). (E) Quantification of DNA damage in intestine (n_WT = 5, n_tert-/- = 5, n_sting-/- = 5, _{tert-/- sting-/-} = 5; WT vs tert-/- p = 0.006, sting-/- vs tert-/- p = 0.008, WT vs tert-/- sting-/- p = 0.007, sting-/- vs tert-/- sting-/- p = 0.0095). (F) Representative western blot images of p53. (G) Quantification of p53 levels in the skin (n_WT = 4, n_tert-/- = 4, n_sting-/- = 4, n_{tert-/- sting-/-} = 4, WT vs tert-/- p = 0.000001, sting-/- vs tert-/- p = 0.000001, tert-/- vs tert-/- sting-/- p = 0.000001). (H) Quantification of p53 levels in the testis (n_WT = 4, n_tert-/- = 4, n_sting-/- = 4, n_{tert-/- sting-/-} = 4, WT vs tert-/- p = 0.050, tert-/- vs tert-/- sting-/- p = 0.048). (I) Quantification of p53 levels in the kidney marrow (n_WT = 4, n_tert-/- = 4, n_sting-/- = 4, n_{tert-/- sting-/-} = 4, sting-/- vs tert-/- p = 0.017, tert-/- vs tert-/- sting-/- p = 0.022). (J) Quantification of p53 levels in the intestine (n_WT = 5, n_tert-/- = 5, n_sting-/- = 5, n_{tert-/- sting-/-} = 5, WT vs tert-/- p = 0.009, sting-/- vs tert-/- p = 0.006, tert-/- vs tert-/- sting-/- p = 0.006). Data were presented as the mean ± s.e.m.; *p < 0.05; **p < 0.01, ***p < 0.001, using a one-way ANOVA and post hoc Tukey test. Source data are available online for this figure.

SASP induced by short telomeres are controlled by the cGAS-STING pathway.

(A) Representative images of senescence-associated beta galactosidase staining in skin, testis, kidney, marrow and intestine (n_WT = 3, n_tert-/- = 3, n_sting-/- = 3, n_{tert-/- sting-/-} = 3). Scale bar = 100 µm. (B) RT-qPCR analysis of inflammatory markers and SASP factors in the skin (n_WT = 4–8, n_tert-/- = 5–8, n_sting-/- = 6–7, n_{tert-/- sting-/-} = 5–7; cdkn2a/b: WT vs tert-/- p = 0.035, sting-/- vs tert-/- p = 0.009, tert-/- vs tert-/- sting -/- p = 0.004; cdnk1a: WT vs tert-/- p = 0.025, sting-/- vs tert-/- p = 0.014, tert-/- vs tert-/- sting-/- p = 0.013; il1b: WT vs tert-/- p = 0.023, sting-/- vs tert-/- p = 0.031, tert-/- vs tert-/- sting-/- p = 0.004,; tgf1b: sting-/- vs tert-/- p = 0.014, tert-/- vs tert-/- sting-/- p = 0.017; mmp15a: sting-/- vs tert-/- p = 0.011, tert-/- vs tert-/- sting-/- p = 0.009). (C) RT-qPCR analysis of inflammatory markers and SASP factors in the testis (n_WT = 4-7, n_tert-/- = 6–11, n_sting-/- = 5–7, n_{tert-/- sting-/-} = 5–8; cdkn2a/b: WT vs tert-/- p = 0.0234; cdnk1a: WT vs tert-/- p = 0.013, tert-/- vs tert-/- sting-/- p = 0.018; il1b: tert-/- vs tert-/- sting-/- p = 0.030; tgf1b: WT vs tert-/- p = 0.029, sting-/- vs tert-/- p = 0.020, tert-/- vs tert-/- sting-/- p = 0.043; mmp15a: WT vs tert-/- p = 0.004, sting-/- vs tert-/- p = 0.016, tert-/- vs tert-/- sting-/- p = 0.028). (D) RT-qPCR analysis of inflammatory markers and SASP factors in the kidney marrow (n_WT = 5–9, n_tert-/- = 5–11, n_sting-/- = 6–11, n_{tert-/- sting-/-} = 6–10; cdkn2a/b: WT vs tert-/- p = 0.0001, sting-/- vs tert-/- p = 0.00003, tert-/- vs tert-/- sting-/- p = 0.0006; cdnk1a: WT vs tert-/- p = 0.002, sting-/- vs tert-/- p = 0.0005, tert-/- vs tert-/- sting-/- p = 0.005; il1b: WT vs tert-/- p = 0.047, sting-/- vs tert-/- p = 0.003, tert-/- vs tert-/- sting-/- p = 0.014; tgf1b: sting-/- vs tert-/- p = 0.005; mmp15a: WT vs tert-/- p = 0.009, sting-/- vs tert-/- p = 0.011, tert-/- vs tert-/- sting-/- p = 0.008). (E) RT-qPCR analysis of inflammatory markers and SASP factors in the intestine (n_WT =  5–7, n_tert-/- = 5–8, n_sting-/- = 5–6, n_{tert-/- sting-/-} = 5–7; cdkn2a/b: WT vs tert-/- p = 0.0004, sting-/- vs tert-/- p = 0.001, tert-/- vs tert-/- sting-/- p = 0.038; cdnk1a: WT vs tert-/- p = 0.007, sting-/- vs tert-/- p = 0.0009, tert-/- vs tert-/- sting-/- p = 0.011; il1b: WT vs tert-/- p = 0.004, sting-/- vs tert-/- p = 0.012, tert-/- vs tert-/- sting-/- p = 0.009; tgf1b: WT vs tert-/- p = 0.034, sting-/- vs tert-/- p = 0.003; mmp15a: WT vs tert-/- NS p = 0.060, sting-/- vs tert-/- p = 0.021, tert-/- vs tert-/- sting-/- p = 0.070). All data are presented as the mean ± s.e.m.; *p < 0.05; **p < 0.01, ***p < 0.001, using a one-way ANOVA and post hoc Tukey test. Source data are available online for this figure.

cGAS-STING pathway control proliferation in telomeric dysfunction.

(A) Representative immunofluorescence images of proliferation and apoptosis. Scale bar = 10 µm. (B) Quantification of proliferation in the skin (n_WT = 5, n_tert-/- = 6, n_sting-/- = 6, n_{tert-/- sting-/-} = 6 WT vs tert-/- p = 0.0001, sting-/- vs tert-/- p = 0.0006, tert-/- vs tert-/- sting-/- p = 0.024). (C) Quantification of proliferation in the testis (n_WT = 6, n_tert-/- = 6, n_sting-/- = 6, n_{tert-/- sting-/-} = 6, WT vs tert-/- p = 0.00007, sting-/- vs tert-/- p = 0.00002, tert-/- vs tert-/- sting-/- p = 0.0006). (D) Quantification of proliferation in the kidney marrow (n_WT = 8, n_tert-/- = 8, n_sting-/- = 6, n_{tert-/- sting-/-} = 8, WT vs tert-/- p = 0.002, sting-/- vs tert-/- p = 0.004). (E) Quantification of proliferation in the intestine (n_WT = 6, n_tert-/- = 5, n_sting-/- = 6, n_{tert-/- sting-/-} = 8). (F) Representative hematoxylin eosin staining of intestine, insets with yellow lines representative of lamina propria thickness. Scale bar = 100 µm. (G) Quantification of lamina propria width (n_WT = 4, n_tert-/- = 4, n_sting-/- = 4, n_{tert-/- sting-/-} = 4, WT vs tert-/- p = 0.042, sting-/- vs tert-/- p = 0.048). (H) RT-qPCR analysis of YAP-TAZ pathway targets in the intestine (n_WT = 4, n_tert-/- = 4, n_sting-/- = 5, n_{tert-/- sting-/-} = 5, ctfg: WT vs tert-/- p = 0.016 sting-/- vs tert-/- p = 0.005, tert-/- vs tert-/- sting-/- p = 0.044; cyr61: WT vs tert-/- p = 0.030, sting-/- vs tert-/- p = 0,023, tert-/- vs tert-/- sting-/- p = 0.046). (I), RT-qPCR analysis of YAP-TAZ pathway targets in the testis (n_WT = 7, n_tert-/- = 8, n_sting-/- = 6–7, _{tert-/- sting-/-} = 7–8, cftg: tert-/- vs tert-/- sting-/- p = 0.008, cyr61: tert-/- vs tert-/- sting-/- p = 0.011). (J) Representative hematoxylin eosin staining of testis. Scale bar = 100 µm. (K) Quantification of mature sperm area (n_WT = 4, n_tert-/- = 4, n_sting-/- = 4, n_{tert-/- sting-/-} = 4, WT vs tert-/- p = 0.0002, sting-/- vs tert-/- p = 0.00007, tert-/- vs tert-/- sting-/- p = 0.006). All data were presented as the mean ± s.e.m.; *p < 0.05; **p < 0.01, ***p < 0.001, using a one-way ANOVA and post hoc Tukey test. Source data are available online for this figure.

cGAS-STING pathway rescues premature aging phenotypes.

(A) Quantification of male fertile capacity (n_WT = 15, n_tert-/- = 10, n_sting-/- = 14, n_{tert-/- sting-/-} = 13), WT vs tert-/- p = 0.000001, sting-/- vs tert-/- p = 0.000001, WT vs tert-/- sting-/- p = 0.011, sting-/- vs tert-/- sting-/- p = 0.0001, tert-/- vs tert-/- sting-/- p = 0.002). (B) Representative images of adult zebrafish and quantification of aging phenotypes scored as kyphosis and cachexia (n_WT = 10, n_tert-/- = 12, n_sting-/- = 11, n_{tert-/- sting-/-} = 12, WT vs tert-/- p = 0.000001, sting-/- vs tert-/- p = 0.000001, WT vs tert-/- sting-/- p = 0.001, sting-/- vs tert-/- sting-/- p = 0.003, tert-/- vs tert-/- sting-/- p = 0.000001). (C) Quantification of seminoma (n_WT = 17, n_tert-/- = 16, n_sting-/- = 21, n_{tert-/- sting-/-} = 18, WT vs tert-/- p = 0.012, sting-/- vs tert-/- p = 0.002, tert-/- vs tert-/- sting-/- p = 0.027). (D) Quantification of weight in adult zebrafish (n_WT = 12, n_tert-/- = 13, n_sting-/- = 11, n_{tert-/- sting-/-} = 13, WT vs tert-/- p = 0.0002, sting-/- vs tert-/- p = 0.004, tert-/- vs tert-/- sting-/- p = 0.012). (E) Quantification of survival n_WT = 24, n_tert-/- = 38, n_sting-/- = 32, n_{tert-/- sting-/-} = 37, WT vs tert-/- p = 0.008, sting-/- vs tert-/- p = 0.005, tert-/- vs tert-/- sting-/- p = 0.010). Data were presented as the mean ± s.e.m. *p < 0.05; **p < 0.01, ***p < 0.001, using a one-way ANOVA and post hoc Tukey test. Seminoma occurrence and survival data were analyzed using log-rank tests, **p < 0.01, ***p < 0.001.