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ATR signaling contributes to cGAS/STING pathway activation in TRF1-deficient MEFs. MEFs were treated with OHT, followed by the ATR inhibitor VE-821 for 24 hours. (A) Detection of micronuclei in MEFs treated with mock (ctr) or ATR inhibitor <t>(ATRi).</t> Cells were fixed and stained with DAPI. Scale bar, 10 μm. Quantification shows the percentage of cells with micronuclei; approximately 100 cells were counted per group. (B-C) Measurement of cellular 2’3’-cGAMP (B) and IFNβ in culture medium (C) by ELISA, with normalization to total protein concentration in the cell pellet. (D) Il6 and Ifnb mRNA levels in OHT-treated MEFs exposed to mock (-) or VE-821 (ATRi), assessed by RT-qPCR. P values were calculated using Student’s t-test (A-C) and one-way ANOVA (D). Data are presented as mean ± SD from three independent experiments for panels B and C, as representative data from one of three independent biological experiments for D.
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ATR signaling contributes to cGAS/STING pathway activation in TRF1-deficient MEFs. MEFs were treated with OHT, followed by the ATR inhibitor VE-821 for 24 hours. (A) Detection of micronuclei in MEFs treated with mock (ctr) or ATR inhibitor (ATRi). Cells were fixed and stained with DAPI. Scale bar, 10 μm. Quantification shows the percentage of cells with micronuclei; approximately 100 cells were counted per group. (B-C) Measurement of cellular 2’3’-cGAMP (B) and IFNβ in culture medium (C) by ELISA, with normalization to total protein concentration in the cell pellet. (D) Il6 and Ifnb mRNA levels in OHT-treated MEFs exposed to mock (-) or VE-821 (ATRi), assessed by RT-qPCR. P values were calculated using Student’s t-test (A-C) and one-way ANOVA (D). Data are presented as mean ± SD from three independent experiments for panels B and C, as representative data from one of three independent biological experiments for D.

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: ATR signaling contributes to cGAS/STING pathway activation in TRF1-deficient MEFs. MEFs were treated with OHT, followed by the ATR inhibitor VE-821 for 24 hours. (A) Detection of micronuclei in MEFs treated with mock (ctr) or ATR inhibitor (ATRi). Cells were fixed and stained with DAPI. Scale bar, 10 μm. Quantification shows the percentage of cells with micronuclei; approximately 100 cells were counted per group. (B-C) Measurement of cellular 2’3’-cGAMP (B) and IFNβ in culture medium (C) by ELISA, with normalization to total protein concentration in the cell pellet. (D) Il6 and Ifnb mRNA levels in OHT-treated MEFs exposed to mock (-) or VE-821 (ATRi), assessed by RT-qPCR. P values were calculated using Student’s t-test (A-C) and one-way ANOVA (D). Data are presented as mean ± SD from three independent experiments for panels B and C, as representative data from one of three independent biological experiments for D.

Article Snippet: The following chemicals were used for cell treatments in the experiments: ATM inhibitor KU-55933 (Abcam, 120637, 10 μM), ATR inhibitor VE-821 (Selleck, S8007, 2.5 μM); cGAS inhibitor RU.521 (Invivogen, inh-ru521, 10 μM), STING-specific inhibitor H-151 (MedChem Express, HY-112693, 7.5 μM), STING ER exit inhibitor Brefeldin A (BFA; Thermo Fisher Scientific, 00-4506-51, 3.0 μg/mL), proteasome inhibitor MG132 (Millipore Sigma, M7449, 5 μM), lysosome inhibitor Bafilomycin A1 (Baf A1; Millipore Sigma, 19-148, 100 nM), and replication stress inducer Aphidicolin (Millipore Sigma, 178273, 0.2 μM).

Techniques: Activation Assay, Staining, Enzyme-linked Immunosorbent Assay, Protein Concentration, Quantitative RT-PCR