FIG 4.

Nrf2 overexpression limited DENV infection. (a) Huh-7 cells were transfected for 24 h with an empty vector (0.5 μg) or an Nrf2 (0.5 μg) or HO-1 (0.5 μg) expression plasmid, infected for 24 h with DENV (MOI, 5), and then evaluated by qPCR analysis for DENV RNA expression levels, which were normalized to GAPDH levels and presented as RNA levels relative to those in uninfected cells, set by default at 1. (b) Cells were evaluated for NRF2, HO-1, and DENV-2 E protein expression by Western blotting (left panel) and were evaluated as described for panel a. DENV infection was detected using an antibody against DENV-2 E protein, and total protein was normalized with an anti-GAPDH antibody; the right panel provides a quantification of DENV-2 E protein levels, expressed as the fold difference over the control. Results are representative of two independent experiments and are expressed as means ± SD. (c) Analysis of DENV RNA expression in Huh-7 cells after pharmacological stimulation of Nrf2 with SFN (20 μM). Cells were treated for 8 h with SFN or DMSO (control) and were then infected for 24 h with DENV (MOI, 5). Viral RNA expression levels were measured by qPCR analysis, normalized to GAPDH levels, and expressed as RNA levels relative to those in uninfected cells, set by default at 1. (d) Quantification of NRF2 and DENV-2 E protein expression by Western blotting (left panel). DENV infection was detected using an antibody against DENV-2 E protein, and total protein was normalized with an anti-GAPDH antibody; the right panel provides a quantification of DENV-2 E protein levels, expressed as the fold difference over the control. Results are representative of two independent experiments and are expressed as means ± SD.