Figure 7. mtDNA Release into the Cytosol Triggers cGAS-STING-Tbk1-Irf3-Mediated Type I Interferon Production.

(A–C) (A) Viability of murine splenocytes treated with ABT-737 ± 20–30 µM Q-VD-OPh (QVD) for 24 hr, (B) caspase activity after 6 hr, and (C) IFN-β in culture supernatant after 24 hr (n = 4 mice per genotype). Means were compared to WT using a one-way ANOVA with Bonferroni correction.

(D–F) (D) Viability of MEFs treated with ABT-737 ± 20–30 µM Q-VD-OPh (QVD) for 24 hr, (E) caspase activity after 6 hr, and (F) IFN-β in culture supernatant after 24 hr (n = 3 independent MEF lines per genotype, or 3 independent CRISPR/Cas9-targeted MEF clones). Means were compared to WT using a one-way ANOVA with Bonferroni correction.

(G–I) (G) Bar graphs of the viability of Mcl1^−/− MEFs pretreated for 1 hr with MRT-67307 (µM) followed by ABT-737 (500 nM) ± 20–30 µM Q-VD-OPh (QVD) for 24 hr, (H) caspase activity after 6 hr, and (I) IFN-β in culture supernatant after 24 hr (n = 3 independent MEF lines).

(J) Immunoprecipitation (IP) followed by PCR. Left, immunoblot of lysates taken from Mcl1^−/− MEFs transduced with an expression plasmid encoding FLAG- cGAS. Right, data represent the fold change (FC) in enrichment of DNA fragments using anti-FLAG or IgG (negative control) to coprecipitate DNA in untreated MEFs or MEFs treated with ABT-737 (1 µM) and Q-VD-OPh (QVD) (30 µM) for the indicated time. DNA fragments were amplified by real-time qPCR using eight primer pairs for mtDNA and two primer pairs for gDNA. The relative locations of the mtDNA amplicons are shown (data are combined from two, with three replicates). Means were compared between treated and untreated samples.

See also Figures S5, S6, and S7.

Unless otherwise stated, means were compared using a two-tailed t test. Data represent the mean ± SEM. *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.005.