PMC

RIG-I localizes to the junctional complex of human colon and small intestine biopsies. RIG-I immunohistochemistry was performed on formalin-fixed paraffin-embedded normal human colon and small intestine biopsies. Black arrows indicate examples of RIG-I association with apical junctional complexes.

RIG-I is relocalized by Rac inhibition and phorbol 12-myristate 13-acetate (PMA) treatment. HEK293 cells were exposed to the specific (A) Rac inhibitor (200 μm, NSC23766) or (B) phorbol 12-myristate 13-acetate (2 μm) for 2 h and then stained for endogenous RIG-I (green) and actin (red).

The CARDs of RIG-I are necessary and sufficient for lamellipodia localization. A, schematic of EGFP-tagged wild-type and mutant RIG-I proteins. Numbers represent amino acid residues in RIG-I. B, confocal micrographs of U2OS cells transfected with EGFP control (EGFP-C2), EGFP-RIG-I, EGFP-ΔCARDs, or EGFP-CARDs and costained for actin (red) 48 h following transfection. C, HEK293 cells were transfected with EGFP-RIG-I, EGFP-ΔCARDs, or EGFP-CARDs and cellular fractionation was performed. Fractions were subjected to immunoblot analysis for GFP (top) and vimentin (below). Data are representative of experiments performed at least three times.

RIG-I regulates cell migration. Wound healing assays were performed in HEK293 cells transfected with EGFP-C2, EGFP-RIG-I, EGFP-Δ-CARDs (A) or with scrambled (control) or RIG-I siRNAs (B). Shown is the percent of wound closure at the indicated times (left) and corresponding representative brightfield images (right). The efficacy of RIG-I siRNA is shown in B. Data are representative of experiments performed at least three times in triplicate. ^*, p ≤ 0.05 (A and B), significant from control cells. Statistical analysis was performed as described under “Experimental Procedures.”

RIG-I localizes to actin-enriched membrane ruffles in HEK293 and U2OS cells. A and B, HEK293 (A) or U2OS (B) cells were fixed and stained with phalloidin to detect actin (red) and with anti-RIG-I goat polyclonal antibody (green). C, HEK293 cells were subjected to cellular fractionation as described under “Experimental Procedures” to obtain cytosolic, membrane/organelle, nuclear, and cytoskeletal fractions. Isolated fractions were subjected to immunoblot analysis for RIG-I (top). Membranes were stripped and reprobed for calpain-2 (middle) and vimentin (bottom) to confirm the purity of cytosolic and cytoskeletal fractions, respectively. D and E, HEK293 cells were transfected with EGFP-RIG-I and fixed and stained for actin (red). F, HEK293 cells were transfected with EGFP-RIG-I and cellular fractionation was performed. Fractions were immunoblotted for GFP (top), calpain-2 (middle), and vimentin (bottom). Data are representative of experiments performed at least three times.

RIG-I associates with apical tight junctions in Caco-2 cells. A, immunofluorescence microscopy for endogenous RIG-I (green) and actin (red) in subconfluent Caco-2 cells grown on collagen-coated glass chamber slides for 24 h. Inset shows ×3 magnification of colocalized (yellow) membrane ruffles. B, confocal microscopy of endogenous RIG-I (green) and ZO-1 (red) in polarized Caco-2 cells cultured for 72 h on collagen-coated glass chamber slides. C, immunofluorescence microscopy for endogenous MDA5 in confluent Caco-2 cells (4′,6-diamidino-2-phenylindole (DAPI) stained nuclei in blue). D, Caco-2 cells grown for 72 h were subjected to cellular fractionation as described under “Experimental Procedures” to obtain cytosolic, membrane/organelle, nuclear, and cytoskeletal fractions. Isolated fractions were subjected to immunoblot analysis for RIG-I (top). Membranes were stripped and reprobed for calpain-2 (middle) and vimentin (bottom) to confirm the purity of cytosolic and cytoskeletal fractions, respectively. E, Caco-2 cells were transfected with EGFP-RIG-I and 48 h later, EGFP-RIG-I localization was assessed. Data are representative of experiments performed at least three times.

Disruption of actin cytoskeleton leads to RIG-I relocalization and the induction of type I interferon signaling. A, immunofluorescence microscopy of RIG-I (green), and actin (red, top) or ZO-1 (red, bottom) in Caco-2 cells exposed to cytoD (8 μm) for 2 h. 4′,6-Diamidino-2-phenylindole (DAPI)-stained nuclei are shown in blue. B, luciferase assays (expressed in relative luminescence activity) from control (No Inh) and cytoD-treated Caco-2 cells transfected with IFNβ and NFκB promoted luciferase constructs. C, Caco-2 cells were treated with vehicle (dimethyl sulfoxide, No Inh) or with cytoD for 2 h and fixed and stained for IRF3 (green) and actin (red). D and E, subconfluent Caco-2 cells cultured for 24 h were exposed to cytoD for 2 h and fixed and stained for RIG-I (green, D) or IRF3 (green, E) and actin (red). F, time-lapse live cell microscopy from EGFP-RIG-I transfected non-confluent Caco-2 cells. Images were taken before and after the addition of cytoD at 10-s intervals. Shown are still images captured at the indicated times following the addition of cytoD. Data are representative of experiments performed at least three times (^*, p ≤ 0.05) (B).