Caspase 4

Cells were lysed in PRO-PREP solution (Intron, Daejeon, Korea). Total protein was measured using BCA protein assay kit (Thermo Scientific, Rockford, IL). Samples were run on sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and transferred onto nitrocellulose membranes. After blocking with 5% skim milk, the membranes were incubated with primary antibodies. Blots were then incubated with peroxidase-conjugated secondary antibodies and visualized by enhanced chemiluminescence (Intron, Daejeon, Korea). The following primary antibodies were used: poly(ADP-ribose) polymerase-1 (PARP-1), activating transcription factor-4 (ATF-4), caspase-4, caspase-12, β-catenin, and α-tubulin (Santa Cruz Biotechnologies, Santa Cruz, CA); CCAAT/enhancer-binding protein homologous protein (CHOP) and caspase-3 (Cell Signaling Technology, Beverly, MA); and β-actin (Sigma-Aldrich, St. Louis, MO).

Commercially available siRNA targeting RIPK1, RIPK3, MLKL, caspase-1, caspase-2, caspase-4, caspase-8 and caspase-10 (Santa Cruz, Dallas, TX) were used to transfect A549 alveolar epithelial cells following the manufacturer instructions. Commercially available CRISPR-Cas9 plasmids to knock out MLKL (Santa Cruz, Dallas, TX) were used following the manufacturer instructions.

Protein assay was performed using the Pierce Rapid Gold BCA Protein Assay kit (Thermo Fisher Scientific) on snap frozen lung lysate or BAL fluid. 25 ug of lysate or equal volumes of BAL fluid (to add 25 ug of the most concentrated sample) were prepared for each of the antibodies to be used in Bolt LDS sample buffer and Bolt sample reducing agent (Thermo Fisher Scientific) and denatured at 70°C for 10 minutes. Samples and SeeBlue Plus2 pre-stained protein ladder were run on Bolt Bis-Tris 4–12% gel (Thermo Fisher Scientific) and transferred to nitrocellulose. Blots were rinsed with water, imaged and de-stained with 1X tris buffered saline with 0.05% Tween 20 (TBS-T). Blots were then blocked with 5% milk and incubated with primary antibody followed by secondary antibody. Immunoblots were exposed to Super Signal West Pico plus or Fempto chemiluminescent substrate (Thermo Fisher Scientific) and exposed to Amersham HyperFilm ECL (Fisher) film for 1 second to 5 minutes and developed using SRX-101A Medical Film Processor (Konica Minolta). The following primary antibodies were used: caspase-4 (Clone: 4B9; Santa Cruz), caspase-8 (90A992; Novus Biologicals); and GSDMDC1 (64-Y; Santa Cruz). Separate gels were run for each of the antibodies using the same sample preparation on the same day with the same loading control.

We purchased antibodies against NLRP3 (AdipoGen; AG-20B-0014-C100), caspase-1 (AdipoGen; AG-20B-0042-C100), caspase-11 (Novus Biologicals; NB-120-10454), IL-1β (R&D Systems; AF-401-NA), caspase-4 (Santa Cruz Biotechnology, Inc; sc-56056), caspase-5 (Santa Cruz Biotechnology, Inc; sc-393346), CREB (Cell Signaling; 48H2), VE-cadherin (Santa Cruz Biotechnology; sc-6458), and β-actin (Sigma; A-5316). Polyclonal anti–SARS-CoV-2 spike glycoprotein was obtained from BEI Resources. IL-1R antagonist anakinra (IL-1RA, #407616) was obtained from Calbiochem; anakinra (C₇₅₉H₁₁₈₆N₂₀₈O₂₃₂S_10.) is a recombinant biopharmaceutical and slightly modified version of the human IL-1RA. Albumin (catalog A7906), O-dianisidine dyhydrochloride (catalog D3252), and protease inhibitor cocktail (catalog P8340) were from Millipore Sigma. The CytoTox 96 Non-Radioactive Cytotoxicity Assay kit (G1780) was purchased from Promega. Enhanced chemiluminescence Western blotting detection reagents and nitrocellulose membranes (Hybond ECL) were from Amersham Biosciences Corp. Lipofectamine 3000 transfection reagents were from Invitrogen.