Fig. 2

The activation of Antioxidant-Responsive Element (ARE)-containing promoter is differentially modulated by ORF6 protein variants. The activity of different SARS-COV-2 ORF6 protein mutants towards the ARE promoter is investigated in (A) A549 and HEK-293T cells transfected with ARE-mediated firefly luciferase (pARE) and SV40 promoter-mediated Renilla luciferase reporter plasmids along with ORF6-expressing plasmids or empty vector (Ctr-). In parallel, (B) transfected HEK-293T cells are vehicle-treated and treated with tert-Butylhydroquinone (tBHQ) or N-acetyl cysteine (NAC) at 36 h post-transfection. A pARE activation reporter assay (C) is conducted in HEK-293T cells over-expressing ectopic NRF2 (FLAG-NRF2) along with ORF6-expressing plasmids or empty vector (Ctr-) and exposed to the proteasome inhibitor MG-132 or to vehicle. At 48 h post-transfection, cells are collected and luciferase activities are measured. Meanwhile, variations in FLAG-NRF2 protein content are assessed by immunoblotting (D) on lysates of vehicle- or MG-132-treated transfected samples. Densitometric analysis is performed and the results are plotted as the mean fold change in target proteins with respect to the empty plasmid-transfected control from three (n = 3) independent experiments ± standard deviations (SD). Significance is determined with respect to the negative control (Ctr-), transfected with empty plasmid, sample as **p < 0.005; *p < 0.05