(A) RIG-I but not MDA-5 confers circRNA sensitivity in HEK293T cells. Luciferase activity of HEK293T cells transfected with plasmids encoding RIG-I or MDA5, along with mouse Ifnb promoter-firefly luciferase/Renilla luciferase vectors, and the indicated circRNA ligands or LMW polyI:C as positive control. Cells transfected with RIG-I or MDA5 only (mock) were used as a negative control. Results are presented relative to Renilla luciferase activity (∗p < 0.05 compared to mock control and for RIG-I over MDA5).

(B) RIG-I is required to sense transfected circRNA in MEFs. Wild-type or RIG-I knockout (KO) MEFs were mock transfected or transfected with 500 ng of linear or circRNA. Relative expression of the indicated mRNA and transfected RNA are measured by qRT-PCR. Means ± SEM are shown (n = 3); ∗p < 0.05 throughout.

(C) RIG-I is required to sense transfected circRNA in HeLa cells. Wild-type or RIG-I knockout (KO) HeLa were mock transfected or transfected with 500 ng of linear or circRNA. Relative expression of the indicated mRNA and transfected RNA are measured by qRT-PCR. Means ± SEM are shown (n = 3). Inset: western blot of RIG-I or actin from wild-type or KO HeLa cells.

(D) CircRNA transfection induces RIG-I foci. HeLa cells were transfected with 500 ng of Cy3-labeled linear or circRNA encoding GFP-IRES and costained for RIG-I. Subcellular distribution of Cy3-labeled RNA (red), RIG-I (detected with secondary antibody conjugated with -Atto647N, pseudo-colored green), and 4′,6′-diamidino-2-phenylindole (DAPI)-stained nuclei (blue) were visualized by confocal microscopy. Scale bar, 2 μm. White arrows indicate areas of RIG-I and RNA colocalization.

(E) Quantification of the RIG-I density. Means ± SEM are shown; n ≈ 41 cells for each condition.

(F) Quantification of the overlap fraction between RIG-I and linear or circRNA. Means ± SEM are shown; n ≈ 41 cells for each condition.