Fig 2: Telomere-specific DNA damage promotes SASP partly via cGAS-STING-TBK1 pathway.a Immunocytochemistry and b quantification of γ-H2AX+, H3K9me3+, or Telomere+ micronuclei (MN—white arrows) in EV and TRF2T188A VSMCs. DAPI (white); H3K9me3 (green), and γ-H2AX (magenta). Magnified upper panel shows H3K9me3 and γ-H2AX colocalizing with MN. Scale bar = 10 μm and 1 μm (inset). c Human VSMCs containing the empty vector (EV) or expressing TRF2T188A stained with DAPI (white) and telo-FISH (yellow). Magnified outlined region shows telo-FISH colocalizing with MN. Scale bar = 10 μm and 2 μm (inset). d Western blot for TBK1 and p65 NF-κB and their phosphorylated forms in EV and TRF2T188A cells. e Relative mRNA expression of selected cytokines in EV or TRF2T188A VSMCs. f Western blot for cGAS, TBK1, p65 NF-κB, and their phosphorylated forms in EV and TRF2T188A cells transfected with non-targeting (NT)- or cGAS-siRNA. g Relative mRNA expression of IL1α, IL8, and CCL20 in EV or TRF2T188A cells transfected with either NT-siRNA or cGAS-siRNA. Data are means±SEM, n = 3–5 independent experiments, ns-non significant (p > 0.05), unpaired, two-tailed Student’s t test or Mann–Whitney U test.

Fig 3: a STING-dependent paclitaxel-induced secretome spreads BCL-xL apoptotic priming.a, b STAT1 and NF-κB pathway analysis of 24 h-paclitaxel treated or not STING−/− (a) and cGAS−/− (b) or control MDA-MB-468 cells by immunoblotting. c Representative pictures of cGAS immunostaining (left panel, scale bar: 20 µm) and quantification of micronucleated cells (middle panel) and cGAS-positive micronuclei (left panel) in control or LMNB2 overexpressing MDA-MB-468 cells after paclitaxel treatment. d cGAS-positive micronucleated cells quantification in paclitaxel-treated or not in PDX #248 and PDX#249 (lower panel) and representative fields after IHC staining with anti-cGAS (upper panel; scale bar: 50 µm). e STAT1 and NF-κB pathway immunoblot analysis in paclitaxel-treated or not, LMNB2 overexpressing or control MDA-MB-468 cells. f 24 h-paclitaxel treated or not (donor) cells were washed out to produce 48 h-conditioned media (CM) that were applied to untreated (recipient) corresponding cancer cells for 48 h in presence or not of the BH3 mimetics WEHI-539, ABT-199, or S63845. Apoptotic index in recipient breast cancer cells was assessed using Annexin-V staining. g–i Same experiment as (f) using control or STING−/− (g) or cGAS−/− (h) or LMNB2 overexpressing (i) indicated donor cells in presence of the BH3 mimetic WEHI-539 or not. j Cell viability of PDX #306 cultured in organoids (BC-PDX#306) in paclitaxel-treated conditions for 4 days and ABT-737 or ABT-199 BH3 mimetics added for the 2 last days (sequential treatment). k Cell viability of 3 distinct patient-derived organoids (BC-01, BC-02, and BC-03) in ABT-737-treated conditions plus paclitaxel or cGAMP. l 24 h-paclitaxel-pretreated control or STING−/− MDA-MB-468 cells were cultured with untreated MDA-MB-468 H2B-RFP expressing cells. After 48 h, co-cultures were treated or not with WEHI-539 for additional 48 h and cell death was assessed in each cell population. Error bars indicate mean +/− SEM; Two-sided paired t-test. The symbols correspond to a p-value inferior to *0.05, **0.01, and ***0.001. NS: not significant. Data were collected from n = 3 independent experiments except d (n = 5).

Fig 4: Scheme of role of AKT1 in regulation of senescence, cGAS-STING, and IKK function. (A) Dividing cell in which CDK4/6 phosphorylation of Rb contributes to E2F-mediated cell cycle progression, and AKT1 promotes cGAS expression. (B) Senescent cell in which Palbociclib (PALBO) inhibits CDK4/6, halting cellular proliferation and contributing to cellular stress and the senescence response through AKT1-mediated NFkB action and cGAS-independent STING activity. Previously undefined abbreviations: CDK = cyclin dependent kinase, Rb = retinoblastoma protein, E2F = E2 transcription factor, ER = endoplasmic reticulum.

Fig 5: The cGAS tethering surface prevents autoreactivity.(A) cGAS KO HeLa cells were reconstituted with the indicated Dox-inducible murine cGAS lentivirus constructs, treated with 0.1 μg /ml Dox for 24 hr, then transfected for four hours with lipofectamine alone or lipofectamine + CT DNA, followed by measurement of cGAMP in cell lysates. (B) Salt elution profiles of WT and R222E mouse cGAS; the top two panels are in HeLa cells and the bottom panel is in TERT-immortalized human fibroblasts. (C) Crystal structures of mouse cGAS (green) assembled on DNA, modeled from PDBID 5N6I. DNA is orange, R222 is highlighted in purple spheres, and the locations of the active sites are noted with red asterisks. (D) Overlay of mouse cGAS (green) and human OASL (cyan; PDBID 4XQ7). The unique loop in cGAS is colored yellow and indicated with the red arrow. (E) Alignments of cGAS across vertebrate phylogeny, with conserved, positively charged residues highlighted in red and the central loop amino acids indicated by the yellow bar. (F) The positively charged residues near R222 are highlighted in purple spheres. (G) Salt elution profiles of cGAS KO HeLa cells transduced with the indicated murine cGAS constructs after induction with 0.1 μg/ml Dox for 24 hr. (H) Cells from (G) were treated with 0.1 μg/ml Dox for 24 hr, then transfected for four hours with lipofectamine alone or lipofectamine + CT-DNA, followed by measurement of cGAMP in cell lysates. (I) Salt elution profiles of cGAS KO HeLa cells transduced with the indicated human cGAS constructs after induction with 0.1 μg/ml Dox for 24 hr. (J) Cells from (I) were treated as in (H), followed by measurement of cGAMP in cell lysates. Statistical comparisons were made between resting WT cGAS and each mutant, using one-way ANOVA of log-transformed biological replicates. *p=0.0155; **p=0.0018; ***p<0.0001; ns: not significant.