Fig. 2

TRIM56 is required for cGAS-dependent signal transduction and acts upstream of STING. a THP-1 cells were stably transfected with control shRNA, TRIM56#1 shRNA, or TRIM56#2 shRNA. Cell lysates were detected with the indicated antibodies. b THP-1 cells used in a were stimulated with HT-DNA (2 μg/ml) and cGAMP (3 μg/ml). The expression of IFNβ mRNA was measured using real-time PCR. c Generation of TRIM56^−/− L929 cells. CRISPR-Cas9 targeting: the CRISPR target site in exon 1 of TRIM56 is underlined, and the protospacer adjacent motif (PAM) is shown in red. Deletions in the three TRIM56 alleles, each of which results in a frameshift, are shown by the black dashes. d cGAS-deleted L929 cells with or without TRIM56 deletion were complemented with wild-type cGAS-FLAG and stimulated with HT-DNA (2 μg/ml) and cGAMP (3 μg/ml). The expression of IFNβ mRNA was measured using real-time PCR. e L929 cell lines used in d were stably transfected with ISREIFNβ-Lucia reporter genes. Cells were stimulated with HT-DNA (2 μg/ml), poly dA:dT (1 μg/ml), poly dG:dC (1 μg/ml) polyI:C (1 μg/ml), and cGAMP (3 μg/ml). Luciferase activity was measured 18 h after stimulation. f L929 cell lines used in e were infected with HSV-1ΔICP34.5 (MOI = 5) and Sendai virus. Luciferase activity was measured 18 h after stimulation. g Schematic representation of the cGAMP bio-assay. Relative cGAMP activity was measured using THP1-Lucia ISG cells that generate luciferase in response to cGAMP. h cGAMP bio-assay. L929 cell lines used in d were stimulated with HT-DNA (2 μg/ml) for 9 h. Extracts of the cells were prepared, DNase- and heat-treated, and incubated with permeabilized THP-1-Lucia ISG cells for 18 h. Relative luciferase activity was measured. Data in a, h are representative of two independent experiments. Data in b, d–f are representative of three independent experiments. Error bars in b, d–f, h indicate mean ± s.d. of n = 3 (b, d–e) and n = 2 (f, h). *P < 0.05, versus control using Student’s t-test (b, d–f, h). Full blots are shown in Supplementary Fig. 10