The tyrosine kinase c-Src is activated by dsRNA, associates with TLR3 and is required for dsRNA-induced IRF-3 and STAT-1 activation. (A) Human mDCs were incubated with dsRNA (100 μg/ml) and activation of c-Src was analyzed by immunoblotting (IB) with an antibody specific to activated phospho-Src. (B) HEK cells expressing Flag-tagged TLR3 were stimulated with dsRNA (100 μg/ml) and lysates were immunoprecipitated (IP) with anti-Flag or mouse IgG and immunoblotted (IB) for the indicated proteins. (C) HEK293 cells were transiently cotransfected with plasmids encoding TLR3 and a luciferase reporter gene containing the Gal4 upstream activation sequence, and expression vectors for Gal4-DBD or Gal4-IRF-3. After 24 h, cells were treated with PP2, PP3, or SU6656 before stimulation with dsRNA. (D) Nuclear extracts were prepared from dsRNA-treated SYF and c-Src-expressing control cells and analyzed by immunoblotting (IB) with an IRF-3 antibody. The blots were reprobed with the nuclear protein XRCC1 as a loading control. (E, F) Determination of IRF-3 localization in c-Src-, Yes-, and Fyn-deficient cells and c-Src-expressing control cells (c-Src) by confocal microscopy. (E) Cells were treated or not with dsRNA and stained intracellularly for IRF-3 (Alexa546). (F) Average percentages of IRF-3 nuclear translocation in SYF cells and c-Src-expressing control cells with nuclear IRF-3 as assessed by confocal microscopy. A total of 200 cells were counted under the different conditions. (G, H) DsRNA-elicited or IFN-β-induced activation of STAT-1 in SYF and c-Src-expressing cells was determined by immunoblotting with an antibody specific to activated STAT-1 (Y⁷⁰¹) and STAT-1. (I) HEK293 cells were transiently cotransfected with vectors encoding TRIF, 0.5, 2, or 20 ng of kinase-inactive c-Src (K297R), and a luciferase reporter gene for IFN-β. Luciferase reporter gene activity was measured after 24 h.

The tyrosine kinase c-Src conveys dsRNA-induced Akt activation. (A) Human mDCs were left untreated or treated with 100 μg/ml dsRNA for various times or 0.5 μg/ml LPS for 20 min. (B) Human mDCs were pretreated or not with PP2 or 25 μM PP3 before addition of 100 μg/ml dsRNA and treatment for 50 min. Cell lysates were immunoblotted (IB) for the indicated proteins. (C) HEK cells expressing Flag-tagged TLR3 were stimulated with dsRNA (100 μg/ml) for the indicated time periods and cell lysates were immunoprecipitated (IP) with anti-Flag, anti-p85, or control IgG and immunoblotted (IB) for the indicated proteins.

DsRNA is internalized in vesicular compartments and traffics to tubular lysosomes. Confocal images of human mDCs incubated with (A) Cy5-labeled dsRNA and (B) Cy5-labeled dsRNA and Alexa543-labeled dextran for 45 min.

DsRNA sequentially colocalizes with early and late endosome marker proteins. (A, B) Confocal images of human mDCs incubated with Cy5-labeled dsRNA as indicated and stained intracellularly for EEA1 (FITC; A) or LAMP1 (Alexa647; B).

DsRNA follows a clathrin-dependent uptake pathway. (A, B) Human mDCs were coincubated with Cy5-labeled dsRNA and transferrin (A) or albumin (B) and analyzed by confocal microscopy. (C) HEK cells expressing CFP-tagged caveolin-1 were incubated with Cy5-labeled dsRNA and analyzed by confocal microscopy. (D) HEK cells were transfected with plasmids encoding E-GFP (60, 50, 40, 20, 0 ng), Eps15DN^GFP (0, 10, 20, 40, 60 ng), Flag-tagged TLR3 (40 ng) and a human IFN-β (40 ng) or an NF-κB (40 ng) luciferase reporter gene. After 36 h, cells were stimulated or not with dsRNA (100 μg/ml) for 6 h and luciferase gene activity was measured. (E) Confocal images of HEK cells expressing Eps15DN^GFP or pEGFP incubated with fluorescently labeled dsRNA.

The tyrosine kinase c-Src colocalizes with dsRNA on endosomes. HEK cells were transfected with c-Src^GFP (A, B) and EEA1^Tomato (A), treated with dsRNA and directly visualized by confocal microscopy (A) or stained intracellularly with LAMP1 (Alexa546; B). (C) Human mDCs were stimulated with Cy5-labeled dsRNA and stained intracellularly with anti-Src (Alexa546) and anti-LAMP1 (FITC) before confocal microscopy.

TLR3 is localized in the ER in resting cells. (A, B) HeLa cells expressing YFP-tagged TLR3 were stained with cholera toxin subunit B (A) or draq 5 (B) and visualized by confocal microscopy. (C–E) HeLa cells expressing TLR3^YFP protein chimera (red) transiently transfected with a CFP-tagged Golgi protein marker (C), a CFP-tagged ER protein marker (D), or stained intracellularly for the ER-resident protein calnexin (E). (F) Untreated human mDCs were permeabilized and stained with anti-TLR3 (TLR3.7; Alexa647) and anti-calnexin (FITC) and examined by confocal microscopy.

TLR3 colocalizes with dsRNA and c-Src on endosomes. (A, B) Confocal images of HeLa cells expressing TLR3^YFP incubated with (A) Cy5-conjugated dsRNA for 10 min or (B) transfected with CFP-tagged FYVE and treated with Cy5-labeled dsRNA for 10 min. (C, D) Human mDCs were treated with dsRNA or not and stained intracellularly for TLR3 (Alexa546) and LAMP1 (Alexa647; C) or TLR3 (Alexa546) and c-Src (Alexa647; D). (E) Whole-cell lysates of HEK cells expressing TLR3^Flag , TLR9^GFP, and TLR4^GFP, and HeLa cells expressing TLR3^YFP were immunoprecipitated with anti-GFP or anti-Flag. Immunoprecipitates were left untreated (−) or were incubated with Endo H (H) or PNGase F (F) and immunoblotted for GFP or TLR3 as indicated.