Figure S3.
SARS-CoV-2 N protein inhibits the activation of NF-κB promoter by TAK1-TAB1/2/3 complex. (A) Schematic diagram of the NF-κB pathway. (B) HEK293 cells were transfected with NF-κB-luc, Flag-empty vector, Flag-SARS-CoV-2 N protein, or Flag-SARS-CoV N protein together with plasmids encoding Flag-RIG-I, Flag-MDA5, Flag-MAVS, Flag-TRAF6, Flag-TAK1, Flag-TAB1, Flag-TAB2, Flag-TAB3, Flag-IKKβ, and Flag-p65. Lysates were collected at 24 h after transfection and luciferase activities were tested. (C) HEK293 cells were transfected with NF-κB-luc, Flag-empty vector, and the increasing amount of Flag-SARS-CoV-2 N protein together with plasmids encoding Flag-MDA5, Flag-MAVS, Flag-TAK1, Flag-TAB1, Flag-TAB2, Flag-TAB3, and Flag-IKKβ. Lysates were collected at 24 h after transfection and luciferase activities were tested. (D) HEK293 cells were transfected with IL-8-luc, Flag-empty vector, and Flag-SARS-CoV-2 N protein together with plasmids encoding Flag-TAK1, Flag-TAB1, Flag-TAB2, and Flag-TAB3. Lysates were collected at 24 h after transfection and luciferase activities were tested. (E) HEK293T cells were transfected with increasing amounts of Flag-SARS-CoV-2 N protein together with HA-TAK1/TAB1/TAB2/TAB3. Lysates were subjected to immunoblot analysis. (F–I) Caco-2 cells stably expressing either SARS-CoV-2 or SARS-CoV N proteins were transfected with siRNA targeting TAB2, p65, or negative control siRNA (siNC). 48 h after transfection, the cells were infected with RDPs for another 48 h. RNA was extracted from the cells for RT-qPCR analysis and lysates were subjected to immunoblot analysis (F and H), or GFP expression was analyzed by microscopy (G and I). Horizontal lines in figures represent the average value of the positive control group. Graphs show mean ± SEM (n = 3 in B–D, F, and H). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant (unpaired, two-tailed Student’s t test). Data in E–I are expressed at least twice in independent experiments, and one representative is shown. Data in B–D are expressed in at least three independent experiments. Source data are available for this figure: SourceData FS3.

SARS-CoV-2 N protein inhibits the activation of NF-κB promoter by TAK1-TAB1/2/3 complex. (A) Schematic diagram of the NF-κB pathway. (B) HEK293 cells were transfected with NF-κB-luc, Flag-empty vector, Flag-SARS-CoV-2 N protein, or Flag-SARS-CoV N protein together with plasmids encoding Flag-RIG-I, Flag-MDA5, Flag-MAVS, Flag-TRAF6, Flag-TAK1, Flag-TAB1, Flag-TAB2, Flag-TAB3, Flag-IKKβ, and Flag-p65. Lysates were collected at 24 h after transfection and luciferase activities were tested. (C) HEK293 cells were transfected with NF-κB-luc, Flag-empty vector, and the increasing amount of Flag-SARS-CoV-2 N protein together with plasmids encoding Flag-MDA5, Flag-MAVS, Flag-TAK1, Flag-TAB1, Flag-TAB2, Flag-TAB3, and Flag-IKKβ. Lysates were collected at 24 h after transfection and luciferase activities were tested. (D) HEK293 cells were transfected with IL-8-luc, Flag-empty vector, and Flag-SARS-CoV-2 N protein together with plasmids encoding Flag-TAK1, Flag-TAB1, Flag-TAB2, and Flag-TAB3. Lysates were collected at 24 h after transfection and luciferase activities were tested. (E) HEK293T cells were transfected with increasing amounts of Flag-SARS-CoV-2 N protein together with HA-TAK1/TAB1/TAB2/TAB3. Lysates were subjected to immunoblot analysis. (F–I) Caco-2 cells stably expressing either SARS-CoV-2 or SARS-CoV N proteins were transfected with siRNA targeting TAB2, p65, or negative control siRNA (siNC). 48 h after transfection, the cells were infected with RDPs for another 48 h. RNA was extracted from the cells for RT-qPCR analysis and lysates were subjected to immunoblot analysis (F and H), or GFP expression was analyzed by microscopy (G and I). Horizontal lines in figures represent the average value of the positive control group. Graphs show mean ± SEM (n = 3 in B–D, F, and H). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant (unpaired, two-tailed Student’s t test). Data in E–I are expressed at least twice in independent experiments, and one representative is shown. Data in B–D are expressed in at least three independent experiments. Source data are available for this figure: SourceData FS3.

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