Sc 393539

Livers were fixed with 4% formalin for 24 h and subsequently embedded in paraffin and cut into 5-μm-thick sections. For liver histopathology, samples were stained with hematoxylin and eosin (H&E). For immunohistochemical (IHC) staining, samples were dehydrated, exposed to antigen, and then incubated with IL-1β (ab283818, Abcam, 1:100 dilution), Caspase 6 (ab185645, Abcam, 1:100 dilution), and Ly-6G (ab261916, Abcam, 1:100 dilution) antibodies respectively at 4 °C overnight. For immune-fluorescence (IF) staining, tissue sections or cultured cells were fixed with 4% formalin for 30 min and then incubated at 4 °C overnight with antibodies against CD11b (ab184308, Abcam, 1:100 dilution), CD68 (#26042, Cell signaling Technology, 1:100 dilution), Caspase 6 (ab185645, Abcam, 1:100 dilution), NR4A1 (ab153914, Abcam, 1:100 dilution); SOX9 (ab185966, Abcam, 1:100 dilution), NEK7 (sc-393539, Santa Cruz Biotechnology, 1:100 dilution), and NLRP3 (ab270449, Abcam, 1:100 dilution). Then samples were incubated with the secondary antibody conjugated to Alexa Fluor 488 (Jackson Immunoresearch) or Alexa Fluor Cy5 (Jackson Immunoresearch) for 2 h at room temperature in the dark. Immunofluorescence images were captured using a fluorescence microscope (Keyence BZ-X810, Osaka, Japan).

Tissues or cells were equilibrated in immunoprecipitation assay buffer at 4 °C for 30 min. The supernatant was collected and centrifuged at 12,000 × g for 20 min. After separating proteins by polyacrylamide gel electrophoresis, they were transferred to polyvinylidene fluoride (PVDF) membranes and incubated with antibodies against Caspase 6 (ab185645, Abcam, 1:1000 dilution), NEK7 (sc-393539, Santa Cruz Biotechnology, 1:1000 dilution), NLRP3 (ab270449, Abcam, 1:1000 dilution), cleaved Caspase 1 (C-caspase 1) (#89332, Cell signaling Technology, 1:1000 dilution), NR4A1 (ab153914, Abcam, 1:1000 dilution), SOX9 (ab185966, Abcam, 1:1000 dilution), S100A9 (ab242945, Abcam, 1:1000 dilution), and β-actin (#4970, Cell signaling Technology, 1:2000 dilution). β-actin was used as an internal reference. The nuclear and cytosolic fractions were prepared with NE-PER Nuclear and Cytoplasmic Extraction Reagents (ThermoFisher Scientific). Lamin B2 (#12255, Cell signaling Technology, 1:1000 dilution) was used as an internal reference of nuclear protein. IBright FL1000 (Invitrogen, Carlsbad, CA, USA) was used to analyze the expression of target proteins. Full and uncropped western blots have been shown in Supplementary Material (2).