Fig. 5

OPTN silencing impairs TBK1 activation after RLR or TLR3 activation. a HEK293T cells were either left unstimulated or infected with Sendai virus (SeV) for 6 and 8 h. Cell lysates (Lys.) were subjected to immunoprecipitation (IP) with an antibody against OPTN. Samples were then analyzed by immunoblotting with antibodies against the indicated proteins. b HEK293T cells were transfected with a control non-specific siRNA (NS) or with two individual OPTN-specific siRNAs (OPTN A and OPTN B) or a NEMO-specific siRNA. The cells were then transfected, 48 h later, with an IFNβ promoter reporter or with a NF-κB reporter and the Renilla luciferase gene as an internal control. Then, 24 h after transfection, cells were either left unstimulated (Unstim) or infected with Sendai virus (+ SeV) for 7 h. Luciferase assays were performed and the results were normalized against Renilla luciferase activity. The data shown are means ± SD from three independent experiments. ****P < 0.0001 versus the NS siRNA-transfected cells (Student’s t test). RLU, relative luminescence units. ns, not significant. c MEFs were transfected with a control non-specific siRNA (NS) or with two individual OPTN-specific siRNAs (OPTN 1 and OPTN 2) or a NEMO-specific siRNA. Then, 72 h later, cells were either left unstimulated or infected with SeV for 6 or 8 h. Cell lysates were analyzed by immunoblotting with antibodies against the indicated proteins. * Indicates non-specific bands. d MEFs were transfected with a control non-specific siRNA (NS) or with two individual OPTN-specific siRNAs (OPTN 1 and OPTN 2). Then, 72 h later, cells were either left untreated (MOCK) or transfected with high molecular weight poly(I:C) (5 μg/mL) for 4 h (trPoly(I:C)). TBK1 aggregation was assessed by immunofluorescence staining and counting of the aggregates. The data shown are means ± SD from three independent experiments (300 cells were counted per condition). **0.001 < P < 0.01 versus the NS siRNA-transfected cells (Student’s t test). e WT or OPTN KO HeLa cells were infected with SeV for the indicated times. Cell lysates were then analyzed by immunoblotting with antibodies against the indicated proteins. * Indicates a non-specific band. f–i WT or OPTN KO HeLa cells were infected with SeV for the indicated times. IFNB1 and IL-6 mRNA levels were then assessed by RT-qPCR with normalization against GAPDH (f, g), or the production of IFNβ and IL-6 was analyzed by ELISA in the cell supernatant (h, i). The data shown are means ± SD from three independent experiments (analysis of variance and comparison with WT HeLa cells in Student’s t test). ns, not significant; AU, arbitrary unit. j HEK293T cells stably expressing TLR3 (HEK293-TLR3) were either left unstimulated or stimulated with poly(I:C) (10 μg/mL) for 30 or 60 minutes. Cell lysates (Lys.) were subjected to immunoprecipitation (IP) with an antibody against OPTN. Samples were then analyzed by immunoblotting with antibodies against the indicated proteins. ° Corresponds to TBK1 detection from a previous immunoblot. k HEK293-TLR3 cells were transfected with a control non-specific siRNA (NS) or with two individual OPTN-specific siRNAs (OPTN A and OPTN B) or a NEMO-specific siRNA. The cells were then transfected, 48 h later, with an IFNβ promoter reporter or with a NF-κB reporter and the Renilla luciferase gene as an internal control. Then, 24 h after transfection, cells were either left unstimulated (Unstim) or stimulated with poly(I:C) (10 μg/mL) for 7 h. Luciferase assays were performed and the results were normalized against Renilla luciferase activity. The data shown are means ± SD from three independent experiments (analysis of variance and comparison with the NS siRNA-transfected cells in Student’s t test). ns, not significant. l HEK293-TLR3 cells were transfected as in (k). Then, 72 h later, cells were either left unstimulated or stimulated with poly(I:C) (10 μg/mL) for 30 or 60 minutes. Cell lysates were then analyzed by immunoblotting with antibodies against the indicated proteins