Nbp2 33422

The expression of GSDMD in isolated AMs was estimated using an immunofluorometric assay. AMs (1 × 10⁶) were cultured on glass coverslips for 24 h, fixed in 4% paraformaldehyde for at least 10 min, and washed three times with PBS. After permeabilization with 0.5% Triton X-100, cells were blocked with 5% BSA (60 min) and incubated overnight on a shaker at 4 °C with rabbit polyclonal anti-GSDMD antibody (1:200; catalog no. NBP2-33422, Novus Biologicals, USA). The sections were then incubated in the dark at room temperature for 1 h with donkey anti-goat IgG antibody conjugated with Alexa Fluor 488 (1:200; Invitrogen, USA). Finally, cell nuclei were stained using 4′,6-diamidino-2-phenylindole (DAPI; Solarbio, China), then viewed under a fluorescence microscope (Olympus BX51).
Image quantification was performed on three randomly selected, non-overlapping fields per section (three sections per mouse) at 100× magnification using an Olympus DP70 microscope (Tokyo, Japan), and the number of pixels per image with an intensity above a predetermined threshold level was quantified using ImageJ software (version 1.47n; National Institutes of Health, Bethesda, MD, USA). The immunoreactivity was expressed as the percentage of total pixels of the imaged field that had an intensity above the threshold level. All quantitative analyses were performed in a blinded manner.

HK‐2 cells and renal tubule tissue were lysed in RIPA buffer (Solarbio, Beijing, China) with protease inhibitor using the following primary antibodies: anti‐TXNIP (14715, Cell Signaling Technology), anti‐NLRP3 (15101, Cell Signaling Technology), anti‐caspase‐1 (24232/3866, Cell Signaling Technology), anti‐cleaved IL‐1β (83186/63124, Cell Signaling Technology), anti‐ASC (sc‐514414, Santa Cruz), anti‐GSDMD (NBP2‐33422, Novus Biologicals, CO/ab219800, Abcam), anti‐cleaved caspase‐1 (4199/89332, Cell Signaling Technology), anti‐RBMX (14794, Cell Signaling Technology), anti‐β‐actin (sc‐81,178, Santa Cruz), and anti‐Histone3 (17168‐1‐AP, Proteintech, Wuhan, China). Western blotting was performed after incubation with a horseradish peroxidase‐conjugated anti‐rabbit/anti‐mouse secondary antibody (D110011/D110087, Sangon Biotech, Shanghai, China).

The following antibodies were used for immunoblotting (dilution; company information): mouse anti-β-actin (1:5000; A2228; Sigma-Aldrich, St. Louis, MI, USA), mouse anti-opsin mAb 4D2 (1:1000; a generous gift from Dr. R. Molday, University of British Columbia, Vancouver, BC, Canada), rabbit anti-gasdermin (GSDRM) (1:1000, NBP2-33422; Novus, Littleton, CO, USA), goat anti-mouse and goat anti-rabbit HRP-conjugated secondary antibody (1:3000; 32430 and 31462; Invitrogen, Carlsbad, CA, USA). Bovine photoreceptor outer segments (OS) were from Invision Bioresources (Seattle, WA, USA).
We independently generated synthetic SDG (referred to as LGM2605 in the literature), as previously described [44 (link)]. Briefly, secoisolariciresinol diglucosides (S,S)-SDG (the major isomer in whole grain flaxseed) and (R,R)-SDG (the minor isomer in whole grain flaxseed) were synthesized from vanillin via secoisolariciresinol and glucosyl donor (perbenzoyl-protected trichloacetimidate under the influence of TMSOTf) through a concise route involving chromatographic separation of diastereomeric diglucoside derivatives (Chemveda Life Sciences Inc., Hyderabad, India). Absorbance spectra of 36mM LGM2605 measured using a SpectraMax M5 fluorescent plate reader (Molecular Devices, San Jose, CA, USA).

Mouse brain tissue was embedded in paraffin and sectioned (3 μm thick), and the sections were subjected to immunofluorescence analysis. After being dewaxed, the sections were blocked with PBS containing 0.3% Triton X-100 and 3% BSA at 37 °C for 1 h. Then, the sections were incubated with rabbit anti-GSDMD (1:100, NBP2-33422, NOVUS, Colorado, USA) and goat anti-CD31 (1:40, AF3628, NOVUS, Colorado, USA) primary antibodies overnight at 4 °C. The next day, the sections were rinsed 3 times with PBS and incubated with the appropriate secondary antibodies (1:100, 711-165-152, Jackson; 1:100, 705-545-003, Jackson, Pennsylvania, USA) for 1 h at room temperature. After being rinsed 3 times with PBS, the samples were stained with DAPI solution. Photographs were taken under a fluorescence microscope. Three sections of each brain tissue were randomly stained, and five random fields of view were photographed.

Immunofluorescence and immunohistochemistry staining were performed on paraffin‐embedded or OCT‐embedded tissues fixed by 4% paraformaldehyde using standard techniques. The primary antibodies were used as followed: anti‐TXNIP antibody (1:200, 14715, Cell Signaling Technology), anti‐NLRP3 (15101, Cell Signaling Technology), anti‐caspase‐1 (24232/3866, Cell Signaling Technology), anti‐ASC (sc‐514414, Santa Cruz), anti‐GSDMD (NBP2‐33422, Novus Biologicals/ab219800, Abcam). The cell nuclei were incubated with 4′,6‐diamidino‐2‐phenylindole (DAPI) (Sigma‐Aldrich). The images were examined using fluorescence microscopy or light microscopy (Olympus, Japan).

Thoracic aortas were isolated, immersed in liquid nitrogen, and then immediately transferred to a −80°C refrigerator until used in western blot assays. Proteins were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and incubated with primary antibodies: eNOS (1 : 1000, ab300071, Abcam), iNOS (1 : 1000, ab283655, Abcam), NOX2 (1 : 2000, 19013-1-AP, ProteinTech), NOX4 (1 : 2000, 14347-1-AP, ProteinTech), p-p65 (1 : 1000, 3033, Cell Signaling Technology), p65 (1 : 1000, 8242, Cell Signaling Technology), NLRP3 (1 : 1000, NBP2-12446, NOVUS), ASC (1 : 1000, sc-514414, Santa Cruz), Caspase-1 (1 : 1000, bs-10743R, Bioss), cGAS (1 : 1000, NBP3-16666, NOVUS), STING (1 : 1000, CST50494, Cell Signaling Technology), GSDMD (1 : 1000, NBP2-33422, NOVUS), Bax (1 : 1000, 50599-2-Ig, ProteinTech), Bcl-2 (1 : 1000, 26593-1-AP, ProteinTech), and β-actin (1 : 5000, 20536-1-AP, ProteinTech). The membranes were then incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG (1 : 5000, ProteinTech) secondary antibodies for 2 h at 37°C and visualized a with chemiluminescent reagent kit (Beyotime, Shanghai, China). The lane densities were read with a Bio-Rad imaging system.

After dewaxing and rehydration, tissue sections were placed in citrate buffer and boiled for 5 min for antigen retrieval, washed twice with PBS, and blocked with 5% goat serum for 30 min. Tissue sections were incubated overnight with primary antibodies overnight at 4°C, washed three with PBS, incubated for 1 h at 37°C with secondary antibodies, and then washed three times with PBS. After diaminobenzidine color development for 5 min, the slides were washed with distilled water to remove float color, and the tissue was counterstained with hematoxylin for 10 s. The tissue was dehydrated in an ethanol gradient with 3 min at each concentration, cleared in xylene for 5 min, mounted with resin, and observed by light microscopy (Leica Inc., Germany). The primary antibodies used were eNOS (1 : 200, ab300071, Abcam), iNOS (1 : 200, ab283655, Abcam), NLRP3 (1 : 200, NBP2-12446, NOVUS), and GSDMD (1 : 200, NBP2-33422, NOVUS). Area quantitative assessment of immunohistochemical staining was performed by the ImageJ 1.8 software (USA).