Fig. 5
From: TRAF3 activates STING-mediated suppression of EV-A71 and target of viral evasion
Silencing TRAF3 expression inhibited cGAS-STING activation and TRAF3 rescued cGAS-STING activation from 2Apro’s suppression. a–e HEK-293T cells were transfected with control siRNA (siNC) or siTRAF3 for 24 h. Next, cells were co-transfected with IRF3 (a), NF-κB (b), IL-8 (c), IFNβ (d) or ISRE (e) promoters, empty vector, or cGAS-STING-expressing vectors. Transactivation of the luciferase reporter gene was determined 18 h after transfection. Luciferase activity induced by cGAS-STING served as a control and was set to 100%. pRL-TK Renilla was used as an internal control. f HEK-293T cells were transfected with control siRNA (siNC) or siTRAF3 for 24 h. Next, cells were co-transfected with empty vector or cGAS-STING-expressing vectors. Eighteen hours post-transfection, cells were harvested for western blot analysis. p-TBK1 (Ser172), p-IRF3 (Ser386), ISG60, TRAF3, p-STING (Ser366), STING and GAPDH were detected using the indicated antibodies. STING was detected by anti-Flag antibody. GAPDH was used as a loading control. g, h Silencing TRAF3 decreased the number and intensity of STING foci formation. HeLa cells were transfected with Control siRNA or siTRAF3. Twenty-four hours later, control siRNA or siTRAF3 cells were transfected with STING-Flag. Sixteen hours post-transfection, cells were primed with 100 nM STING agonist or solvent control. Six hours post agonist addition, cells were fixed, treated with Triton-X100, and incubated in 5% FBS, and subcultured to anti-Flag and following FITC Conjugate Goat Anti-Rabbit IgG (H + L) reactivation. Images were captured using a ZEISS laser scanning confocal microscope (Zeiss LSM 900, Oberkochen, Germany). ZEISS ZEN Microscope software was used for acquisition. Percentages of foci formation cells were statistical analyzed. i Co-immunoprecipitation analysis of endogenous TRAF3 with STING-Flag. HEK-293T cells were transfected with STING-Flag or a control vector, as indicated. Cell lysates were prepared and immunoprecipitated using anti-Flag beads 36 h after transfection. Precipitated samples were separated by SDS-PAGE, transferred to polyvinylidene fluoride membranes, and reacted with anti-TRAF3 antibodies to detect potential binding of TRAF3 protein and anti-Flag antibodies to detect STING-Flag. GAPDH was used as a loading control. j The effect of knocking down TRAF3 on STING-TBK1 interaction. HEK-293T cells were transfected with control siRNA (siNC) or siTRAF3. Twenty-four hours later, HEK-293T cells were co-transfected with cGAS and STING-Flag expression vectors or control vectors for 16 h. Cell lysates were prepared and immunoprecipitated using anti-Flag beads. Precipitated samples were separated by SDS-PAGE, transferred to polyvinylidene fluoride membranes, and reacted with anti-Flag, anti-TBK1, anti-p-TBK1 (Ser172), anti-TRAF3, and anti-ISG60 antibodies to detect indicating proteins. GAPDH was used as a loading control. k–p HEK-293T cells were transfected with IRF3 (k), NF-κB (l), IL-8 (m), IFNβ (n), or ISRE (o) promoter and cGAS-STING expressing vectors, with empty vector or EV-A71 2Apro WT or EV-A71 2Apro C110A expression vectors, in the presence or absence of vectors expressing TRAF3, as indicated. Transactivation of the luciferase reporter gene was determined 18 h after transfection. Luciferase activity induced by cGAS-STING served as a control and was set to 100%. pRL-TK Renilla was used as an internal control. p HEK-293T cells were transfected with cGAS-STING-expressing vectors, empty vector, or EV-A71 2Apro in the presence or absence of vectors expressing TRAF3, as indicated. Eighteen hours post-transfection, cells were harvested for western blot analysis. Cell lysates were separated by SDS-PAGE, transferred to PVDF membranes, and treated with antibodies to detect p-IRF3 (Ser386), ISG60, cGAS, and STING. Anti-Flag antibody was used to detect TRAF3. Anti-HA antibody was used to detect EV-A71 2Apro. GAPDH was used as a loading control. Data in b–m represent the average of results from three independent experiments (n = 3, representative immunoblots are shown). The error bars indicate the standard deviations of data from three independent experiments. Means and standard deviations are presented. Statistical significance was determined by two-sided unpaired Student’s t-test, **p < 0.01; ***p < 0.001; ****p < 0.0001
