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. 2020 Jul 29;10(21):9644–9662. doi: 10.7150/thno.47651

Figure 2.

Figure 2

The cytosolic DNA sensor cGAS is activated after cerebral ischemia/reperfusion. (A) The mRNA level of cGAS in primary microglia isolated from brain tissue 3 h post-tMCAO (9 mice per group). (B) The protein level of cGAS in primary microglia isolated from brain tissue at 1, 2, 3 and 6 h post-tMCAO, respectively. The gray values were analyzed by using ImageJ and were normalized to Iba1. (C) BV2 cells were transfected with siRNA against cGAS and harvested 48 h post-transfection to quantify the mRNA level of cGAS. (D) cGAS-silenced and control BV2 cells were transfected with poly(dA:dT) and harvested 3 h post-poly(dA:dT) treatment to analyze the protein levels of pIRF3, cGAS, and GAPDH. (E) cGAS-silenced, STING-silenced, and control BV2 cells were transfected with poly(dA:dT) and harvested 6 h post-poly(dA:dT) treatment to analyze the mRNA level of IFN-β. (F) BV2 cells were transfected with siRNA against STING and harvested 48 h post-transfection to quantify the mRNA level of STING. (G) cGAS-silenced, STING-silenced, and control BV2 cells were transfected with mtDNA and harvested 6 h post-transfection to analyze the mRNA level of IFN-β. (H) The cGAS-mtDNA complex was isolated from brain tissue extracted from mice subjected to tMCAO using an anti-cGAS antibody and quantified by real-time PCR. (I) The cGAS-mtDNA complex was isolated from OGD/R-treated primary microglia using an anti-cGAS antibody and quantified by real-time PCR with primer pairs against mtDNA. (J and K) The mRNA levels of IFN-β (J) and IL-6 (K) in control and cGAS-silenced BV2 cells after OGD/R treatment. (* indicates p < 0.05, ** indicates p < 0.01 by ANOVA or Student's t-test). Abbreviations: cGAS, cyclic GMP-AMP synthase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IFN-β, interferon-beta; IL-6, interleukin-6; mtDNA, mitochondrial DNA; OGD/R, oxygen-glucose deprivation/reperfusion; pIRF3, phosphorylated interferon regulatory factor 3; siRNA, small interfering RNA; STING, stimulator of interferon genes; tMCAO, transient middle cerebral artery occlusion.