Journal: bioRxiv

Article Title: sTelomere replication stress-induced DNA damage response triggers inflammatory signaling via canonical and non-canonical STING pathways

doi: 10.1101/2025.07.11.664434

Figure Lengend Snippet: TRF1 depletion leads to STING ubiquitination and activates the NFκB pathway via cGAS and ATM. (A) MEFs were treated with OHT for the indicated times. Immunoprecipitation (IP) was performed on cell lysates using K63-linked polyubiquitin-specific antibody-conjugated Dynabeads. Input (left panel) and IP (right panel) samples were analyzed by Western blot with the indicated antibodies. IgG was used as a control (with limited exposure). Black dashes on the left indicate STING protein. (B) STING knockdown MEFs were treated with OHT, followed by the ATM inhibitor KU-55933 for 24 hours. IP was performed as described in (A). Input (left panel) and IP (right panel) samples were analyzed by Western blot. Black dashes on the left indicate STING protein. (C) Western blot analysis of p65 levels in nuclear and cytosolic fractions and whole-cell lysates from MEFs treated with mock (-) or OHT (+). A longer exposure of p65 is shown in the lower panel. Black dashes on the left indicate the protein of interest. (D) Immunofluorescence analysis of p65 (green) showing nuclear localization in OHT-treated MEFs. Nuclei were counterstained with DAPI (blue). Scale bar, 40 μm. (E) Transcriptional DNA binding activity of nuclear p65 measured in nuclear extracts from MEFs treated with mock (-) or OHT (+). (F-H) Il6 mRNA levels were assessed by RT-qPCR: (F) in MEFs treated with mock (ctr) or OHT for the indicated times; (G) in MEFs treated with OHT for 24 hours, followed by ATM inhibitor KU-55933 (ATMi) for 72 hours; (H) in MEFs treated with OHT for 24 hours, followed by cGAS inhibitor (cGASi) for 48 hours. P values were calculated using Student’s t-test (E), one-way ANOVA (G, H), and two-way ANOVA (F) for comparisons. Data are presented as mean ± SD from three independent experiments for (E) and from a representative experiment of three independent experiments (F–H).

Article Snippet: For immunoprecipitation, approximately 50 μL of protein G or protein A Dynabeads (Invitrogen, Thermo Fisher Scientific, 10003D and 10008D) were incubated with either K63-linked specific polyubiquitin antibody (Cell Signaling Technology, 5621) or STING antibody (Cell Signaling Technology, 50494) at 4°C for 1 hour with rotation to allow antibody binding.

Techniques: Ubiquitin Proteomics, Immunoprecipitation, Western Blot, Control, Knockdown, Immunofluorescence, Binding Assay, Activity Assay, Quantitative RT-PCR