Fig. 1.

SARS-CoV-2 S-RBD and Orf3a induce pyroptosis and HMGB1 release in lung cells (a) Transfection with SARS-CoV-2 viral proteins induces BEAS-2B lung cell death (cell death was assessed after 24 h of transfection). Graph of qRT-PCR fold change values shows the efficacy of transfection. (b) Western blots demonstrating increased IL-1β and HMGB1 levels in BEAS-2B cells transfected with SARS-CoV-2 S-RBD and Orf3a. Blots are representative of data from three independent experiments with similar results. Blots were stripped and re-probed with β-actin to establish equivalent loading. (c) Increase in absorbance at 405 nm representing induced active caspase-1 in SARS-CoV-2 S-RBD and Orf3a-transfected BEAS-2B cells. Increase in caspase-1 activity is accompanied by HMGB1 release as demonstrated by Western blot using cell culture supernatant. (d) Treatment with 1 mM glycyrrhizin diminishes SARS-CoV-2 S-RBD and Orf3a-induced caspase-1 activity. Inset shows the efficacy of glycyrrhizin in inhibiting HMGB1 release. Ponceau S staining of the blots was used to establish equivalent loading in case of supernatants. (e) Increase in absorbance representing rescue of cell death by HMGB1 inhibitor Glycyrrhizin. (cell death was assessed 48 h post transfection) One-way ANOVA (with Tukey’s multiple comparison post-hoc test) was used for statistical analysis. p < 0.05*, p < 0.01**, p < 0.001***