Figure 4: DENV/DENV-ADE-induced RLR activation mediates NOS2 expression independent of IFN signalling.

K562 cells was stably transfected with plasmid expressing RIG-I W/WT MDA-5. All the cells were subjected to DENV and DENV-ADE infection. (a) Cell lysates were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and stained with specific antibodies against RIG-I, MDA-5, iκ-B, NF-κB and NOS2 6 h post-inoculation. N = 3. (b) Cells were harvested for total RNA 48 h post-inoculation; the intracellular viral RNA was detected by qRT-PCR, with GAPDH as an internal control. The fold enhancement was expressed as the ratio of DENV/DENV-ADE. N = 3; error bars show the means ± SEM. K562 cells was transfected with siRNA target MAVS or siRNA control. (c) cell lysates were subjected to detection of MAVS and NOS2 4 h post-infection. (d) Total cellular RNA was extracted for intracellular viral RNA quantitation 48 h post-inoculation. (e) K562 cells were co-transfected with pLR-TK plasmid (internal control) and pGL3 -hIFNβ or pGL3-ISRE plasmid (experimental reporter) and then subjected to DENV or DENV-ADE infection. At the indicated time points post-infection, reporter gene activity was measured by a dual-luciferase reporter assay. N = 3; error bars show the means ± SEM. (f) K562 cells were infected with DENV alone, DENV-antibody complex, or mock infected. Cellular RNA was extracted and used for gene expression quantification of IFNγ and IFNλ using qRT-PCR at indicated time points. N = 3; error bars show the means ± SEM.