Cell lines and cell culture
Mouse mononuclear RAW264.7 macrophages were purchased from ATCC (Manassas, VA, USA) and cultured in Dulbecco's Modified Eagle's medium supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA, USA) at 37°C and 10% CO2. LPS (Sigma-Aldrich, St. Louis, MO, USA) or recombinant mouse HMGB1 protein (Novus Biologicals, Centennial, CO, USA) was added into the medium to stimulate RAW264.7 cells at the indicated concentration. 5 mM ATP was added for 1 hour before subsequent experiments.
Western blot analysis
Soluble protein in the culture supernatant was precipitated with 7.2% trichloroacetic acid plus 0.15% sodium deoxycholate. Cells were lysed on ice in RIPA lysis buffer (Thermo Fisher Scientific) supplemented with a protease inhibitor cocktail (Thermo Fisher Scientific). Equal amounts of protein (30 µg) were then separated by 10% − 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes. The membranes were blocked with 5% fat-free dry milk in Tris Buffered Saline with Tween 20 for 1 h and incubated with primary antibodies, including anti-IL-1β (Cell Signaling Technology, Danvers, MA, USA) and anti-caspase-1 (Santa Cruz Biotech, Dallas, TX, USA) for soluble proteins, and anti-IL-1β, anti-caspase-1, anti-LC3, anti-HMGB1, anti-GAPDH (all from Santa Cruz Biotech) and anti-Gasdermin D (GSDMD) (Abcam, Cambridge, UK) for cytoplasmic proteins for 14 h at 4°C. The blots were washed and incubated with HRP-conjugated secondary antibodies (Santa Cruz Biotech) for 1 h at room temperature. Protein signals were visualized using an ECL kit (Thermo Fisher Scientific), followed by imaging using the Bio-Rad imaging system (Bio-Rad, Hercules, CA, USA).
Caspase-1 activity analyses
Caspase-1 activity was determined using the Caspase 1 Activity Assay Kit (Beyotime, China) following the manufacturer’s instructions. Briefly, 50 µg of total cytosolic protein was incubated with 20 nmol Ac-YVAD-pNA overnight at 37°C. Caspase-1 activity was evaluated by the production of pNA, which was determined by measuring absorbance at 405 nm using a spectrophotometer (Thermo Fisher Scientific).
Flow cytometry analysis of cell pyroptosis
Cells were incubated with FAM-labeled caspase-1 FLICA (Bio-Rad, Hercules, CA, USA) at 37°C for 1 h. Cells were fixed with 4% paraformaldehyde and then stained with TMR red-labeled In-Situ Cell Death Detection reagent (Roche Applied Science, Indianapolis, IN, USA) for 1 h. The cells were analyzed by flow cytometry using a FACScalibur flow cytometer (BD Biosciences, San Jose, CA, USA). Background and auto-fluorescence were determined using isotype controls. The double-stained cells were counted as pyroptotic cells, and the rate of pyroptosis was calculated.
Cell transfection
RAW264.7 cells were cultured in 12-well plates for 24 h before transfection. Cells were transfected with HMGB1 shRNA lentiviral particles (Santa Cruz Biotechnology) using Polybrene. After 48 h of transfection, the efficiency of HMGB1 knockdown was confirmed by real-time PCR and western blotting.
Real-time PCR
Total RNA was isolated using the Trizol reagent (Ambion, USA). cDNA was prepared using a Verso cDNA synthesis kit (Thermo Fisher Scientific) following the manufacturer’s protocol with a total reaction volume of 20 µl. Real-time PCR was performed using a SYBR green master mix qPCR kit (Thermo Fisher Scientific) on the ABI 7500 real-time PCR system. The primers for HMGB1 were 5′-GCTGACAAGGCTCGTTATGAA-3′ (forward) and 5′ -CCTTTGATTTTGGGGCGGTA-3′ (reverse), and those for GAPDH were 5′ -AGGTCGGTGTGAACGGATTTG-3′ (forward) and 5′ -GGGGTCGTTGATGGCAACA-3′ (reverse). Relative mRNA expression was normalized to GAPDH using the 2−ΔΔCt method.
Immunofluorescence confocal microscopy
RAW264.7 cells were cultured on glass coverslips in 12-well plates and treated with anacardic acid (AA, Sigma-Aldrich) (25 mmol/l) or anti-HMGB1 antibody (10 µg/ml) for 12 h. Then, the coverslips were fixed with 2% paraformaldehyde for 15 min at room temperature and permeabilized with 0.1% Triton X-100 for 10 min at room temperature. After incubation with anti-HMGB1 antibody (1:500) overnight at 4°C, cells were incubated with Alexa Fluor Plus 555 conjugated secondary antibody (Thermo Fisher Scientific) (1:200) for 1 h. 4',6-diamidino-2-phenylindole was used for nuclear staining. Images were acquired with a confocal microscope (Zeiss LSM 510 Meta; Carl Zeiss).
Statistical analysis
The data from at least three experiments are presented as the mean ± SD. The significance of differences between multiple groups and between two groups was determined using one-way ANOVA followed by Tukey’s post-hoc test and the two-tailed Student's t-test, respectively. P < 0.05 was considered statistically significant.