Figure 1: Tollip-deficiency impairs STING-mediated IFN signaling.

(A and B) Arrayed siRNA screen of 21 different gene targets in MEFs. Quantitative RT-PCR (qRT-PCR) expression of Ifnb mRNA following cGAMP stimulation (2 μg/ml, transfected by Lipofectamine, same below) for 5 h (A) or intracellular polyI:C (1 μg/ml, transfected by Lipofectamine, same below) for 5 h (B). A representative experiment with 2 biological replicates is showing (same below). Experiments were repeated for at least three times (same below).

(C) qRT-PCR analysis of Ifnb mRNA following cGAMP stimulation for 5 h. Wild type MEFs were treated with control siRNA (siCtrl) with 4 distinct siRNA sequences targeting Tollip for 48 h before cGAMP stimulation. Right panel shows that cells with greater Tollip KD had less Ifnb mRNA expression.

(D) qRT-PCR analysis of Ifnb mRNA in Tollip KD MEFs stimulated with cGAMP over a 24 h time course.

(E) qRT-PCR analysis of Ifnb mRNA in Tollip KD MEFs stimulated with increasing amount of cGAMP (0, 2, 3, 5 μg/ml) for 5 h.

(F) qRT-PCR analysis of Ifnb, Il6 mRNA and several ISGs in Tollip KD MEFs stimulated with STING agonist DMXAA (50 μg/ml) for 2 h. Heat map summarizes qRT-PCR data on ISG expression.

(G) qRT-PCR analysis of Ifnb in Tollip KD cells stably expressing an empty vector or siRNA-resistant Flag-TOLLIP. Cells were stimulated with DMXAA (50 μg/ml) for 2 h.

(H and I) Tollip^+/+ or Tollip^−/− MEFs were stimulated with increasing amount of DMXAA (0, 1, 10, 100 μg/ml) for 2 h (H) or intracellular polyI:C (0, 0.01, 0.1, 1.0, 5.0, 10.0 ng/ml) for 5 h (I). Ifnb mRNA was analyzed by qRT-PCR

(J) Bone marrow-derived macrophages (BMDMs) from Tollip^+/+ or Tollip^−/− mice were stimulated with dsDNA (1 μg/ml), cGAMP (2 μg/ml) or DMXAA (50 μg/ml) and Ifnb mRNA was measured by qRT-PCR. For panel G, ***, p < 0.001; n.s., not significant. Error bars represent the SEM. Unpaired Student’s t test (2-sided).

Data are representative of at least three independent experiments.