Fig. 1

SARS-CoV-2 ORF6 protein induces cellular stress. A549 cells ectopically expressing wild-type (wt)-ORF6 or the M₅₈R and D61 mutants are stained with the reduced, nonfluorescent form of fluorescein (H₂DCFDA) as an indicator for Reactive Oxygen Species (ROS). Upon cleavage of the acetate groups by intracellular esterases and oxidation, the highly fluorescent 2',7'-dichlorofluorescein (DCF) is determined by (A) fluorescence microscopy and (B) by microplate reding (Ex:485/Em:535) on cell lysates. The results are presented as the mean fold change in fluorescence intensities ± standard deviations (SD) from at least three (n > 3) independent experiments, each condition tested in duplicate. The same samples are analysed for (C) reduced glutathione (GSH) levels. The differences between total GSH and its oxidized form (GSSG), normalized to the protein content of each sample, are calculated. The results are provided as the mean fold change in GSH content ± standard deviation (SD) from three (n = 3) independent experiments. Significance is determined with respect to the negative control (Ctr-), transfected with empty plasmid, sample as p < 0.005, ^**; p < 0.05, ^*; or to the wt-ORF6 variant as p < 0.05, ^#;$, p < 0.005, ^$$