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. 2022 Feb 22;50(5):2681–2699. doi: 10.1093/nar/gkac079

Figure 7.

Figure 7.

cGAS is activated in response to a major fraction of micronuclear DNA in NBS1- and CtIP-depleted cells. (A) Schematics show tripartite cGAS system consisting of G10-Flag-cGAS, cGAS-HA-G11 and GFP1-9 for detecting activated cGAS in cells. The principle is that when two different molecules of cGAS, i.e., G10-Flag-cGAS and cGAS-HA-G11, dimerize on double-stranded DNA, that binds to GFP1-9, which results in a functional GFP. (B) Western blots show detection of G10-Flag-cGAS and cGAS-HA-G11 expression in HEK 293 cells. Cells were infected with lentiviral particles carrying G10-Flag-cGAS, cGAS-HA-G11 and GFP1-9, then were selected with Puromycin, G418 and hygromycin. Total cell lysate was probed with anti-HA, anti-Flag-HRP, cGAS and β-actin antibodies. (C, D) cGAS is activated in a major fraction of micronuclear DNA in NBS1 and CtIP knockdown cells. Representative images show GFP fluorescence signal in the micronuclei triggered by the dimerized (i.e. activated) cGAS (C). Bar graph shows the percentages of activated cGAS-positive micronuclei in shSCR, shNBS1 and shCtIP cells at 24 h after 2.5 Gy ionizing radiation (IR) and at 72 h after 1 μM 6-thio-dG treatment (D). HEK293 cells stably expressing tripartite cGAS were transfected with shSCR, shNBS1 and shCtIP RNAs and treated with either 2.5 Gy IR or 1 μM 6-thio-dG, and the GFP signal in the micronuclei was analyzed at indicated times after the treatment. Bar graph presents the mean and STDEV from 300–400 cells in three independent experimental groups. Statistical analysis was performed using Student's t-test. (E) Co-depleting cGAS and NBS1, and cGAS and CtIP abrogates cellular senescence. Bar graph shows percentages of β-gal–positive cells expressing SCR, NBS1 alone, CtIP alone, cGAS alone, NBS1+cGAS and CtIP+cGAS shRNAs at 10 days after 3 μM 6-thio-dG (6-dG) treatment. Bar graph presents the mean and STDEV of three independent experiments. Statistical analysis was performed using Student's t-test. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001. (F) Model depicting the mechanism of NBS1-mediated regulation of cGAS binding to micronuclei and the subsequent activation of immune signaling, which culminates in cellular senescence.