Fig. 2

EV-A71 infection inhibits cGAS-STING pathway activation. a HEK-293T cells were infected with EV-A71 (MOI of 0.5) or mock-infected with virus preservation solution. Twenty-two hours later, cells were transfected with IRF3, NF-κB, IL-8, IFNβ, or ISRE promoter, together with cGAS-STING expression vectors or empty vector. Eighteen hours post-transfection, cells were harvested for western blot analysis. Cell lysates were separated by SDS-PAGE, transferred to PVDF membranes, and incubated with the indicated antibodies to detect p-TBK1 (Ser172), p-IRF3 (Ser386), ISG60, ISG56, ISG54, RSAD2, EV-A71 VP1, p-STING (Ser366), ISG15 and GAPDH. Anti-myc and anti-Flag antibodies were used to detect cGAS and STING expression. GAPDH was used as a loading control. b–f. cGAS-STING-stimulated promoters were suppressed following EV-A71 infection. Eighteen hours post transfection (a), IRF3 (b), NF-κB (c), IL-8 (d), IFN-β (e), and ISRE (f) promoter luciferase activity was measured using a dual-luciferase reporter gene assay. Luciferase induced by cGAS-STING served as a control and was set to 100%. pRL-TK Renilla was used as an internal control. g HEK-293T cells were infected with EV-A71 (MOI of 0.5) or mock-infected with virus preservation solution. Twenty-two hours later, cells were transfected with cGAS-STING expression vectors or empty vector. Eighteen hours post-transfection, cells were harvested for total RNA extraction. First strand cDNA synthesis and RT-qPCR analysis with indicated interferon stimulated genes specific primers. Human genes activated by cGAS-STING transfection served as a positive control and were set to 100%. Total RNA was prepared from 293 T cells and analyzed for the transcriptional level of the indicated genes by RT-qPCR. Data in a–g represent the average of results from three independent experiments (n = 3, representative immunoblots are shown). Error bars indicate the standard deviation of the data from three independent experiments. The means and standard deviations are presented. Statistical significance was determined using a two-sided unpaired Student’s t-test, *p < 0.05; **p < 0.01; ***p < 0.001