Fig 1: Autophagy role in NSP5/ORF3a-mediated p62 modulation. (a–d) THP-1 transfected with control plasmid or plasmids encoding NSP5/ORF3a were incubated in cell culture medium with 5% FBS, in the presence or absence of lysosomal inhibitor bafilomycin A1 (BAF; 50 nm) during the last 6 h of 48 h incubation period. Intracellular LC3-II and p62 were detected by immunoblotting (a,b), and their levels were quantified by densitometry and expressed relative to ß-actin as a loading control (c,d). Representative blots are shown in (a,b), while the data from four independent experiments (c,d) are presented as fold change relative to the control value (dashed line) obtained in cells transfected with control plasmid and not treated with BAF (median and 25/75 percentile values are shown as lines; * p < 0.05, two-tailed one-sample t-test vs. untreated control; # p < 0.05, two-tailed paired t-test). Original blots from (a,b) are shown in Supplementary Figure S11.
Fig 2: NSP5 and ORF3a regulate cytokine expression through p62. (a–c) THP-1 cells were transfected with control plasmid or plasmids encoding NSP5/ORF3a (a), control/p62 siRNA (b), or control/LC3 siRNA (c). After 48 h, the cytokine (TNF, IL-1β, IL-6, IL-10, IL-33) mRNA levels were measured by RT-qPCR (a–c). The downregulation of p62 and LC3-II was confirmed by immunoblotting (b,c). The data from six (a) or five (b,c) independent experiments are presented as fold change relative to the control value (dashed line) obtained in cells transfected with control plasmid/siRNA (median and 25/75 percentile values are shown as lines; * p < 0.05, two-tailed one-sample t-test). Original blots from (b,c) are shown in Supplementary Figure S13.
Fig 3: p62 levels are reduced in COVID-19. The levels of autophagy markers p62, LC3, and ATG5 in the blood plasma of control subjects (n = 19) and COVID-19 patients (n = 26) at hospital admission (day 0) and 7 days later were determined by ELISA (median and 25/75 percentile values are shown as lines; * p < 0.05, two-tailed Mann–Whitney U test or Wilcoxon signed-rank test for controls vs. COVID-19 patients or COVID-19 patients at day 0 vs. day 7, respectively).
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