Af 401 na

Flagellin purified from P. aeruginosa was purchased from InvivoGen. DOTAP liposomal transfection reagent was purchased from Sigma-Aldrich. Lipopolysaccharide (LPS), ATP, nigericin, pyocyanin, and proteinase K were purchased from Sigma-Aldrich. N-3-oxo-dodecanoyl-l-homoserine lactone (3-oxo-C12-HSL) and N-butyryl-l-homoserine lactone (C4-HSL) were obtained from Cayman. Antibodies were acquired to detect mouse caspase-1 (Adipogen, 20B-0042), mouse IL-1β (R&D, AF-401-NA), mouse IL-6 (Cell Signaling, 12912), mouse NLRP3 (Adipogen, 20B-0014), mouse ASC (Santa Cruz, SC-22514-R), and Flagellin (InvivoGen, mabg-flapa).

Human renal TECs and epithelial cell medium were obtained from ScienCell. Iohexol was obtained from GE Healthcare and Mannitol was obtained from Sigma. Mouse anti-rabbit IgG and Mouse anti-rat IgG antibodies were purchased from Cell Signaling Technology. We obtained antibodies targeting caspase-11 (Sigma, clone 17D9), caspase-4 (Abcam., ab40887), caspase-5 (Abcam, ab25898), IL-1β (R&D Systems, AF-401-NA), GAPDH (Cell Signaling Technology, #2118), Gsdmd (Novus Biologicals, NBP2-33422), and cytokeratin-18 (Abcam, ab668). Pan-caspase inhibitor Z-VAD-FMK was obtained from R&D Systems.

Ultrapure LPS and nigericin were purchased from Invivogen and Sigma-Aldrich, respectively. The following antibodies were purchased commercially: rabbit anti-NLRP3 (cryo-2; Adipogen, AG-20B-0014), rabbit anti-ASC (AL177; Adipogen, AG-25B-0006), mouse anti-caspase-1 (p20) (Casper-1; Adipogen, AG-20B0042), mouse anti-caspase-1 (p10) (Casper-2; Adipogen, AG-20B0044), goat anti-mouse IL-1β antibody (R&D, AF-401-NA), anti-gasdermin D (Cell Signaling, #93709), anti-actin (clone 4; Merck Millipore, #MAB1501), anti-F4/80 (Cell Signaling, #70076).

Protein extraction from macrophages was performed using TRIzol reagent according to the manufacturer's instructions. Briefly, after phase separation using chloroform, 100% ethanol was added to the interphase/phenol–chloroform layer to precipitate genomic DNA. Subsequently, the phenol–ethanol supernatant was mixed with isopropanol to isolate proteins. The Bradford method was used to determine protein concentrations in cell lysates. Equal amounts of protein or supernatants were separated by 13.5% SDS–PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane. Membranes were incubated overnight with antibodies against CASP11 (Cell Signaling, 14340), murine CASP1 (Adipogen, AG‐20B‐0042‐C100), human cleaved CASP1 (Cell Signaling Technology, 4199), human CASP1 (Cell Signaling Technology, 2225), cleaved CASP7 (Cell Signaling Technology, 9491), human CASP4 (MBL, M0293), murine IL‐1β (R&D Systems, AF‐401‐NA), and GAPDH (Cell Signaling Technology, 2118). Corresponding secondary antibodies conjugated with horseradish peroxidase and in combination with enhanced chemiluminescence reagent were used to visualize protein bands. Densitometry analyses were performed by normalizing target protein bands to their respective loading control (GAPDH) using ImageJ software as previously described 8, 9.