Additional file 3 of O-GlcNAc of STING mediates antiviral innate immunity

Additional file 3. Figure S1. (A). Immunoblotting of phosphorylated IRF3 (p-IRF3), IRF3, OGT, and actin in shCtrl and shOGT cells infected with ISD (2 μg/mL) for 16 hrs. (B). Immunoblotting of phosphorylated IRF3 (p-IRF3), IRF3, OGT, and actin in shCtrl and shOGT cells infected with poly(dA:dT) (2 μg/mL) for 16 hrs. Data are representatives from 3 independent experiments. Figure S2. (A). O-GlcNAcylated proteins in KYSE-30 cells were pulled down with sWGA beads. cGAS was detected with an anti-cGAS antibody from abcam. (B). Immunoblotting of K63 ubiquitination in immunoprecipitated complex pulled down with STING antibody from lysates of cells treated with or without DON (10 μM) for 12 hrs. STING was immunoprecipitated with anti-STING antibody from abcam. OGT, and STING in the pulldown complex and in the input were detected with immunoblotting. (C). STING was immunoprecipitated with anti-STING antibody from abcam. K63-Ub, OGT, and STING in the pulldown complex and in the input were detected with immunoblotting. (D). HEK-293T cells were transfected with FLAG-tagged STING-WT or -T229A. Co-IP was performed with an anti-STING antibody from abcam. Immunoblotting was performed with antibodies against K27-Ub, K63-Ub, RL2, TRIM56 and STING. Data are representatives from 3 independent experiments. Figure S3. (A). Analyses of STING oligomerization by native gel electrophoresis. cGAMP (9 μg/mL, 16 h) was used to induce high-order oligomerization of STING. The results shown are representatives of three biological repeats. (B). KYSE-30 cells were transfected with FLAG-tagged STING-WT or -T229A and treated with 2 μg/mL ISD for 16 hrs. Cells were then fixed for immunofluorescence detection of STING and calnexin (ER marker) or GM130 (Golgi marker). Data are representatives from 3 independent experiments. Figure S4. KYSE-30 cells reconstituted with either STING-WT or STING-T229E were transfected with 2 μg/mL poly(dA:dT) by Lipofectamine 2000 for 16 hrs. Levels of Ifnb1, Il6, Tnfa, Isg15, Cxcl10, and Mx1 mRNA in cells were measured with RT-PCR. Data from representatives of 3 independent biological replicates are presented. Data are means ± SEM. ** p < 0.001, *** p < 0.0001. Figure S5. (A-C). Detection of HSV-1 in the liver, lung, and kidney from WT and Sting1-/- mice challenged with HSV-1 (5 x 106 PFU/mouse) for 6 days (n = 6). (D). Detection of liver tissue injury in WT and Sting1-/- mice pretreated with or without DON and challenged with either PBS or HSV-1 (5 x 106 PFU/mouse) for 6 days (n = 6 per group), scale bar = 100 µm. (E). Detection of kidney tissue injury in WT and Sting1-/- mice pretreated with or without DON and challenged with either PBS or HSV-1 (5 x 106 PFU/mouse) for 6 days (n = 6 per group), scale bar = 100 µm. Data from representatives of 3 independent biological replicates are presented. Data are means ± SEM. NS, p > 0.05, *** p < 0.0001. Figure S6. Immunohistochemical staining was applied for the detection of STING and total O-GlcNAc modification in lungs from WT and Sting1-/- mice pretreated with or without DON and challenged with either PBS or HSV-1 (5 x 106 PFU/mouse) for 6 days (n = 6 per group), scale bar = 100 µm. Data are representatives of 3 independent biological replicates. Data are means ± SEM. NS, p > 0.05, * p < 0.05, **p < 0.001, *** p < 0.0001. Figure S7. Immunohistochemical staining was applied for the detection of STING and total O-GlcNAc modification in livers from WT and Sting1-/- mice pretreated with or without DON and challenged with either PBS or HSV-1 (5 x 106 PFU/mouse) for 6 days (n = 6 per group). Data are representatives of 3 independent biological replicates. Data are means ± SEM. NS, p > 0.05, * p < 0.05, ** p < 0.001, *** p < 0.0001.