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

Figure 6.

Figure 6

Deficiency of HDAC3 in microglia suppresses the cGAS-STING-mediated autoimmune response. (A and B) Adult primary microglial cells were isolated from HDAC3 cKO (n = 6) and WT (n = 6) mice, and the mRNA levels of HDAC3 (A) and cGAS (B) were analyzed by real-time PCR. (C) The neurological deficits of HDAC3 cKO (n = 7) and WT (n = 15) mice subjected to tMCAO were assessed 24 h post-reperfusion. (D and E) Brain tissue isolated from WT (n = 14) and HDAC3 cKO (n = 7) mice 24 h post-reperfusion was stained with triphenyl tetrazolium chloride, and infarct volume was determined using ImageJ (D) and infarct volume was determined using Image J (E). (F) RNA was extracted from the ischemic penumbra cortex and contra cortex of HDAC3 cKO (n = 12) and WT (n = 14) mice subjected to tMCAO 6 h post-reperfusion, and the mRNA level of IL-6 was quantified by real-time PCR. (G) Serum was isolated from HDAC3 cKO (n = 13) and WT (n = 21) mice subjected to tMCAO 6 h post-reperfusion, and the level of IL-6 was quantified by ELISA. (H to J) The number (H and J) and soma area (I and J) of Iba1+ cells in the ischemic penumbra and contralateral cortex of HDAC3 cKO and WT mice subjected to tMCAO 24 h post-reperfusion. (K) Proteins extracted from the ischemic penumbra and contralateral cortex of HDAC3 cKO and WT mice subjected to tMCAO 6 h post-reperfusion were analyzed with antibodies against cGAS, HDAC3, Iba1, and GAPDH. (L and M) Mice administered RGFP966 (n = 11) or vehicle (n = 5) for 2 days underwent tMCAO, and infarct volume was determined by TTC staining. (N) Serum was isolated from RGFP966-treated (n = 8) and vehicle-treated (n = 6) mice subjected to tMCAO 6 h post-reperfusion and the level of IL-6 was quantified by ELISA. (* indicates p < 0.05, ** indicates p < 0.01 by ANOVA or Student's t-test). Abbreviations: cGAS, cyclic GMP-AMP synthase; cKO, conditional knockout; ELISA, enzyme-linked immunosorbent assay; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HDAC3, histone deacetylase 3; Iba1, ionized calcium-binding adapter molecule 1; IL-6, interleukin-6; STING, stimulator of interferon genes; tMCAO, transient middle cerebral artery occlusion; TTC, triphenyl tetrazolium chloride; WT, wild-type.