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. 2019 Jul 25;116(33):16479–16488. doi: 10.1073/pnas.1901090116

Fig. 4.

Fig. 4.

TMEM203 levels regulate TBK1/IRF3 activation downstream of STING. (A) CRISPR/Cas9-mediated Tmem203 knockout results in reduced TBK1 and IRF3 phosphorylation upon STING stimulation. Vector or CRISPR/Cas9-mediated Tmem203 knockout (KO Tmem203) RAW 264.7 cells were stimulated with 3′3′-cGAMP (1 µg/mL) for the time indicated. Activation of TBK1 and IRF3 was examined by Western blot analysis. Membranes were blotted with anti-phospho-TBK1 (p-TBK1), anti-total TBK1 (t-TBK1), anti-phospho-IRF3 (p-IRF3), and anti-total IRF3 (t-IRF3) as indicated (n = 3). (B) Increased TBK1-IRF3 activation in Tmem203-overexpressing RAW 264.7 cells in response to STING stimulation. Vector- or Tmem203-overexpressing (OE Tmem203) RAW 264.7 cells were stimulated with 3′3′-cGAMP (1 µg/mL) for the time indicated. Activation of TBK1 and IRF3 was examined by Western blot analysis. Membranes were blotted with anti-phospho-TBK1 (p-TBK1), anti-total TBK1 (t-TBK1), anti-phospho-IRF3 (p-IRF3), and anti-total IRF3 (t-IRF3) as indicated (n = 3). (C) Increased TBK1-IRF3 activation in Tmem203-overexpressing RAW 264.7 cells in response to HSV infection. Vector- or Tmem203-overexpressing RAW 264.7 cells were infected with HSV-1 for the time indicated. Activation of TBK1 and IRF3 was examined by Western blot analysis. Membranes were blotted with anti-phospho-TBK1 (p-TBK1), anti-total TBK1 (t-TBK1), anti-phospho-IRF3 (p-IRF3), and anti-total IRF3 (t-IRF3) as indicated (n = 3). (D) IFNβ cytokine secretion is impaired in Tmem203 knockout RAW 264.7 cells (Left) and is enhanced in Tmem203-overexpressing cells (Right) in response to HSV infection. CRISPR/Cas9-mediated Tmem203 knockout (or Vector) and Tmem203-overexpressing (or Vector) RAW 264.7 cells were infected with HSV-1 for 6 h, and Ifnb1 secretion was measured by ELISA. n = 3. (E and F) Tmem203 overexpression enhances IRF3 nuclear accumulation in macrophages in response to STING stimulation. Vector- and Tmem203-overexpressing RAW 264.7 cells were stimulated with 1 µg/mL 3′3′-cGAMP for 6 h. Subcellular localization of IRF3 was determined by IRF3 intracellular staining, and confocal fluorescence images were captured (F) (Scale bar: 10 µm). Average IRF3 nuclear fluorescence intensity of 50 to 200 cells was quantified using ImageJ (F). n = 3. *P < 0.05, **P < 0.01, ***P < 0.001.