DNase I treatment or PAD4 deficiency inhibits tPA-induced upregulation of cGAS-STING and type 1 IFN signaling. (A-B) Representative immunoblots and quantification of cGAS expression in the ischemic cortex 24 hours after stroke in WT mice treated with vehicle, tPA, or tPA in combination with DNase I and in WT and Pad4^−/− mice treated with tPA compared with mice undergoing sham surgery (n = 5). (C) Representative immunoblots for STING, phosphorylated (pTBK1) and total TBK1, and pIRF3 and total IRF3 expression in the ischemic cortex. (D-F) Quantification of band intensities for each group (n = 5). (G-H) Quantification of IFN-β and IL-6 levels by enzyme-linked immunosorbent assay in the ischemic cortex (n = 6). (I-J) Double immunostaining of cGAS and STING with microglial cells (Iba1), macrophages (RM0029-11H3), and neutrophils (Ly6G) in mice subjected to MCAO and tPA treatment. Scale bar, 20 μm. (K-L) Quantification of activated microglia and macrophage infiltration in the ischemic cortex (n = 6). (M-O) Quantification of the numbers of pIRF3⁺, IFN-β⁺, and IL-6⁺ microglial cells (Iba1) in the ischemic cortex (n = 6). Values are means ± standard deviation. *P < .05. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NS, not significant.