Ab242945

The liver tissues were removed and fully lysed using a RIPA lysis buffer. The homogenates were centrifuged at 13,300 rpm at 4 °C for 15 min. The protein concentrations in the supernatant were quantified by the BCA method (Thermo Fisher, Carlsbad, CA, USA). A total of 50 μg of protein per sample was resolved by 7.5–15% SDS–PAGE and transferred onto a PVDF membrane. Next, the membranes were blocked in 5% skim milk for 1 h at room temperature and incubated with the appropriate primary antibodies at 4 °C overnight, and then incubated with the secondary antibodies at room temperature for 1 h. Finally, the membranes were developed on a FluorChem M System (PerkinElmer, Waltham, MA, USA). The intensities of protein bands were quantified with NIH Image J version 1.48, and the relative levels of the corresponding proteins were calculated in each group. The primary antibody against S100A9 (ab242945, 1:1000) was from Abcam (Cambridge, MA, USA). Antibodies against S100A8 (47310; 1:1000), p-AMPK (2535; 1:1000), AMPK (5831; 1:1000), p-AKT (4060; 1:2000), AKT (4691; 1:1000), ACC (3662; 1:1000) and β-actin (4970; 1:1000) were from Cell Signaling Technology (CST, Danvers, MA, USA). Antibodies against GLUT4 (66846-1-Ig; 1:3000), Bax (50399-2-Ig; 1:7000) and Bcl-2 (12789-1-AP; 1:2000) were obtained from Proteintech Group (Chicago, IL, USA).

LC3B (ab48394, Abcam, 1:1000), p62 (ab56416, Abcam, 1:1000), S100a9 (ab242945, Abcam, 1:1000), HIF-1ɑ (CPA9305, Cohesion Biosciences), Atg9a (ab108338, Abcam), β-actin (81115-1-RR, Proteintech), and GAPDH (bsm-33033M, Bioss) were used for western blot. S100a9 (26992-1-AP, Proteintech) and HIF-1ɑ (sc-13515, Santa Cruz) were used for immunofluorescence.

The primary antibodies used for western blotting included: MMP9 (ab228402, Abcam, USA), β-actin (RM2001, Beijing Ray, Beijing, China).
The primary antibodies used for IHC included: S100A9 (ab242945, Abcam), Ly6G (ab238132, Abcam), CD11b (ab133357, Abcam), F4/80 (70,076 S, CST, Danvers, MA, USA), IL-1β (12,242 S, CST), MMP9 (ab228402, abcam).
The primary antibodies used for immunofluorescence (IF) included: CD11b (ab133357, Abcam), F4/80 (70,076 S, CST), IL-1β (12,242 S, CST), MMP9 (ab228402, abcam), SPC (ab211326, abcam).
The primary antibodies used for flow cytometry included: TruStain FcX anti-mouse CD16/CD32 antibody (Biolegend, 101,319), FITC anti-mouse/human CD11b (Biolgend, 101,205), PE/Cyanine7 anti-mouse Ly-6G (Biolegend, 127,617), Alexa Fluor 647 Rat anti-mouse S100A9 (BD Pharmingen, 565,833) and PE anti-mouse IL-1β (Thermo, 12-7114-80).
Other reagents included: Cultrex Basement Membrane Extract (R&D, Minneapolis, MN, USA); recombinant murine IL-1β (Peprotech, Rocky Hill, USA), murine SAA3 (General Biol, China); IKK 16, JNK-IN-7, Losmapimod and Ravoxertinib (MedChemExpress, Monmouth Junction, NJ, USA).

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).