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. 2020 Sep 4;9:e57837. doi: 10.7554/eLife.57837

Figure 4. ROS regulate interferon response by inhibiting STING dimerization.

(A) BMDMs were treated with vehicle or 200 μM H2O2 for 10 min in serum free culture medium, then stimulated with 1 μg/ml DMXAA. Western blots of TBK1, IRF3, STING, pTBK1 and pIRF3 were performed at 0 min, 30 min, 60 min and 90 min after stimulation. Data shown are representative of 2 independent experiments. (B) BMDMs were treated with vehicle or different concentrations of H2O2 in serum free culture medium for 10 mins, then stimulated with 1 μg/ml DMXAA. Level of pTBK1 was determined at 60 min after stimulation. n = 1 (C) BMDMs were treated with vehicle or 25 μM menadione for 30 min, then stimulated with 1 μg/ml DMXAA. Western blot of TBK1, IRF3, STING, pTBK1 and pIRF3 was performed at 0 min, 30 min, 60 min and 90 min after stimulation. Data shown are representative results of two independent experiments. (D) BMDMs isolated from WT control or Stinggt/gt mice were treated with vehicle or 8 μM menadione for 16 hr, then infected with MHV68 at MOI = 5. Virus titer was determined by plaque assay at 0 hr, 10 hr, 24 hr, 48 hr and 72 hr after infection. n = 3 with three technical repeats each time. (E) BMDMs were treated with vehicle, 25 μM menadione, 25 μM menadione and 2 mM NAC for 30 min, then stimulated with 1 μg/ml DMXAA. STING polymerization was determined by non-reducing SDS-PAGE. M: STING monomer; D: STING dimer. Data shown are representative of 2 experiments.

Figure 4.

Figure 4—figure supplement 1. Schematic diagrams of STING monomer, dimer and polymer on different electrophoresis gels.

Figure 4—figure supplement 1.

STING is a dimeric protein at resting state, which is formed by non-covalent bonds and can be disrupted by SDS treatment. Ligand binding alters the conformation of STING and triggers its oligomerization through the formation of covalent disulfide bonds. These disulfide bonds are resistant SDS treatment but sensitive to reducing reagents. As such, STING proteins that are manifested as monomer and dimer on non-reducing SDS PAGE gels are actually dimer and oligomer, respectively.
Figure 4—figure supplement 2. ROS decrease TBK1 recruitment during STING activation.

Figure 4—figure supplement 2.

BMDMs were treated with vehicle or 25 μm menadione for 30 min, then stimulated with 1 μg/ml DMXAA for 1 hr. Cells were lysed and immunoprecipitated with anti-STING antibody, and the protein levels of TBK1 and STING in both total cell lysate and immunoprecipitated samples were determined by western blot.