Figure 2.

SARS-CoV-2 Nsp5 enhances cytokine expression through activating NF-κB signaling pathway. (A) 293T cells were co-transfected with the reporter vectors of NF-κB or AP-1 with Flag-Nsp5 or empty vector. Cells were harvested after transfection for 36 h and assayed for dual-luciferase activity. (B) Calu-3 cells were transfected with Flag-Nsp5 or empty vector for 36 h, the cytosol and nucleus fractions of cells were prepared. The protein p65, Flag-Nsp5, Lamin B (loading control of nucleus) and Actin (loading control of cytoplasm) were analyzed by western blot. The relative expression levels of p65 in nucleus to p65 in cytoplasm were estimated by densitometry calculation. (C–E) Calu-3 cells were transfected with an empty or Flag-Nsp5 vector for 36 h and treated with inhibitor MLN120B for 12 h before cells were harvested with a final concentration of 5 or 10 μM. Then, cells were immuno-stained with anti-p50 antibody (green) and counterstained with Hoest33342 to examine chromosomes (blue) (C). Expression of p50 protein in cytosolic and nuclear were detected by western blot and the relative expression levels of p50 in nucleus to p50 in cytoplasm were estimated by densitometry calculation (D). mRNA levels of IL-1β and IL-6 measured with qRT-PCR (E). Data are presented as Mean ± SEM for three biological replicates, and statistical significance was calculated by one-way ANOVA, **P < 0.01, ***P < 0.001, NS, not significant.