Caspase 1

The sterilized coverslips were placed into the wells of a 24-well plate, and then RIMVECs were added to each plate at the concentration of 1 × 10⁵ cells/well in 600 μL DMEM. When the cells reached 90% confluence onto the coverslip, they were divided into the same groups as described in the in vitro experiment paragraph. At the end of the incubation time, the cells were fixed with 500 μL paraformaldehyde (PFA) for 40 min, followed by permeabilization with 0.2–0.5% Triton X-100 for 20 min. The cells were washed three times with phosphate buffered saline (PBS) before and after each step and then incubated with the following primary antibodies at 4°C overnight: NLRP3 (1:1000, ABclonal), GSDMD (1:1000, ABclonal), caspase-1 (1:1000, ABclonal), and ZO-1 (1:1000, Affinity Biosciences). Next, the secondary antibody (1:5000 dilution in 5% BSA, ABclonal) was added, and the cells were incubated at room temperature for 1 h in the dark. One μg/mL 4’,6-diamidino-2-phenylindole (DAPI) was added to each well, and the cells were incubated at room temperature for 20 min in the dark. After washing, the coverslips were removed from the wells and blotted to remove any excess water. Each coverslip was placed onto a microscope slide, and the slides were sealed with nail polish and observed under a confocal fluorescence microscope (NIKON Ti-S, Tokyo, Japan).

The expression of proteins, including NLRP3 and caspase-1 in HG-induced NRK-52E cells, was assessed through immunofluorescence analysis [15 (link)]. Cells grown on a coverslip fixed in a 6-well plate were simultaneously incubated with BCA (5 μM, 10 μM, and 20 μM) and HG (30 mmol/L) in serum-free media. After 48 h of incubation, cells were treated with paraformaldehyde (4%), followed by permeabilization (Triton X-100 0.2%), and blocking with normal goat serum (5%). The cells were then washed and probed with primary antibodies NLRP3 (1:150 dilution, ABclonal Inc., Woburn, MA, USA) and caspase-1 (1:150 dilution, ABclonal Inc., Woburn, MA, USA), followed by washing and incubation with Alexa fluor 488 or 594 labelled anti-rabbit secondary antibody (Invitrogen, Waltham, MA, USA) at room temperature for 1 h. The nuclear stain was undertaken through DAPI stain (ThermoFisher Scientific, Waltham, MA, USA). The cells were visualized under a confocal microscope (Leica TCS SP8).

Protein from pancreatic tissue and INS-1 cells lysates were collected. Western blot procedures were processed as standard protocol with specific antibodies against TXNIP (1:1,000, Abcam, UK), GSDMD (1:1,000, Abclonal, USA), caspase-1 (1:1,000, Abclonal, USA), NLRP3 (1:1,000, Adipogen, USA), and β-actin (1:5,000, Sigma, USA). The blots were visualized by ChemiDoc™ Touch Imaging System (Bio-Rad, CA, USA). Western blot image was analyzed and quantified by using Image J software (NIH, Bethesda, MD, USA). Relative expression of the protein was normalized with β-actin expression.

Cell culture medium and human primary renal PTECs were provided by ScienCell (San Diego, CA, United States). Anti-SLC2A9 (URAT1) antibodies, TSG101, CD63, caspase-1, GSDMD, IL-1β, caspase-3, caspase-8, caspase-9, cytochrome c, Bcl-2, and Bax were purchased from ABclonal (Wuhan, China). Oxonic acid (OA) and UA were provided by Sigma (St. Louis, MO, United States). CIAS1/NALP3, GAPDH, and anti-GLUT9 were provided by Abcam (Cambridge, United Kingdom). In addition, the CCK-8 assay kit was provided by Jiwei Biological Technology (Shanghai, China).

Heart tissues were lysed in radioimmunoprecipitation assay (RIPA) buffer (Beyotime, Shanghai, China) containing protease and phosphatase inhibitors. The protein concentrations were determined using the BCA assay (CWBiotech, Beijing, China); 40 μg of total proteins were loaded per lane, separated using 10% sodium dodecyl sulfate (SDS-PAGE), and blotted onto nitrocellulose membranes. The membrane was incubated overnight with the following primary antibodies: AMPK, p-AMPK, Atg5 and BECN1 (1:1,000, Cell Signaling Technology, USA), Bcl-2, Bax, caspase-3 and β-actin (1:1,000, Proteintech, China), IL-1β, mTOR, and p-mTOR (1:1,000, Abcam, UK), NLRP3, ASC and caspase-1 (1:1,000, Abclonal, China). The membranes were washed and incubated with the corresponding secondary antibodies. Finally, the bands were visualized using an ECL Kit (CW0049, CWBIO, Beijing, China).

Western blot analysis was performed according to a previously described standard method23 (link). Primary antibodies against the following targets were used for the Western blot analyses: HER2 (#2165, diluted at 1: 500, Cell Signaling Technology), Cyclin B1 (#4138, 1: 1000, Cell Signaling Technology), Cyclin D1 (#2922, 1: 1000, Cell Signaling Technology), HMGB1 (ab79823, 1: 10000, Abcam), GSDMB (A7474, 1: 1000, ABclonal), caspase-1 (A18646, 1: 1000, ABclonal), NLRP3 (A12694, 1: 1000, ABclonal), ERK (#9102, 1: 1000, Cell Signaling Technology), phosphorylated (p)-ERK (#4370, 1: 1000, Cell Signaling Technology), AKT (#9272, 1: 1000, Cell Signaling Technology), phosphorylated (p)-AKT (#4060, diluted at 1: 1000, Cell Signaling Technology), Nrf2 (#12721, 1: 1000, Cell Signaling Technology), β-actin (used as the loading control; sc-47778, Santa Cruz Biotechnology), GAPDH (used as the loading control; sc-47724, Santa Cruz Biotechnology), and β-tubulin (used as the loading control; #2128, Cell Signaling Technology).