Gsdmd

The cell proteins were extracted by using a Membrane and Cytosol Protein Extraction Kit (Beyotime Biotechnology, Shanghai, China). Then the proteins were then quantified using a Pierce^™ Microplate BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, United States). A total of 20 μg proteins were separated on a 10% sodium dodecyl sulfate-polyacrylamide gel and then transferred onto polyvinylidene difluoride membranes (Millipore, Bedford, MA, United States). All membranes were cultured overnight at 4°C with primary antibodies against NLRP3 (Affinity), caspase-1 (Affinity), gasdermin d (GSDMD, Affinity), IL-1β (Affinity), IL-18 (Affinity) and tubulin (Affinity) after blockage by 5% skim milk for 1 h. Then the membranes were incubated with appropriate secondary antibodies (Affinity) at room temperature for 1 h. Finally, the blots were detected by using an ECL Western Blotting Substrate (Affinity) and FluorChem R detection system (ProteinSimple, United States).

MDP [C₂₉H₃₂O₁₃S, molecular weight: 620], purity > 98%, was provided by the Chemistry Laboratory of the Institute of Clinical Pharmacology of Anhui Medical University (Hefei, China); LW6 (CAS: 934593-90-5) was obtained from MedChemExpress (USA). N-acetyl-L-cysteine (NAC) was purchased from Beyotime Biotechnology (Shanghai, China). Dulbecco's modified Eagle's medium (DMEM) was obtained from Gibco Co. Ltd. (CA, USA). Fetal calf serum was purchased from Wisent Co. Ltd. (Canada). Streptomycin, penicillin, ROS Assay Kit, and goat anti-rabbit IgG were purchased from Beyotime Biotechnology Co. Ltd. (Shanghai, China). Enzyme-linked immunosorbent assay (ELISA) kits for IL-1β and IL-18 were the products from J&L Biological (Shanghai, China). Antibodies against GRK2, p-GRK2 S670, HIF-1α, NLRP3, ASC, Caspase-1, GSDMD, and β-actin were purchased from Affinity Bioscience (Taiwan, China). The antibody against VHL was the product from Wanleibio (Shenyang, China).

The ovarian tissues were fixed in 4% paraformaldehyde solution, then dehydrated and embedded in paraffin. The tissues were sectioned at 5 µm. The slides were dewaxed in xylene and rehydrated through graded ethanol concentrations. Antigen retrieval was performed in citrate solution and then were blocked with 3% BSA for 1 h. After incubating with the primary antibody (CD3, Affinity Biosciences, DF6594; CD14, ABclonal, A19011; DDX4, Abcam, ab27591, GSDMD, Affinity, AF4012) overnight at 4 °C, the slides were incubated with appropriate secondary antibody (CY3 goat anti‐mouse IgG, EarthOx, E031610‐01; CY3 goat anti‐rabbit IgG,EarthOx, E031620‐01; DyLight 488 goat anti‐rabbit IgG, EarthOx, E032220‐01; Alexa Fluor 647 goat anti‐mouse, Abcam, ab150115) for 1 h at room temperature, followed by staining with 4′,6‐diamidino‐2‐phenylindole (DAPI, meilunbio) and imaged with Leica TCS‐SP8 Confocal Laser Scanning Microscope (Leica Microsystems).

The steps of protein extraction and Western blot analysis are referred to the previous literature.¹⁵ In brief, the extracted bladder protein tissue was loaded into a 10% SDS‐polyacrylamide gel and transferred into a PVDF membrane after electrophoresis. After incubating with primary and secondary antibodies and washing (NLRP3, ASC, Pro‐caspase‐1, Caspsae‐1 p20, GSDMD, GSDMD‐N and Cleave‐IL‐1β, Affinity; Goat Anti‐Rabbit IgG (H + L) HRP, Affinity), the ECL Plus assay Kit (Affinity, K002) was used for colour rendering. The gel analysis system scanned each strip protein, and the grey value of the strip was measured by image analysis software (Image J).

Rat primary chondrocytes were sonicated in radioimmunoprecipitation assay (RIPA) lysis buffer (Gibco, Grand Island, NY, USA). The protein concentration of different groups should be normalized. The protein was separated by electrophoresis and blocked by 5% skim milk after transmembrane. After that, the membranes were incubated overnight with primary antibodies targeting MYD88 (Abcam), matrix metallopeptidase 3 (MMP3, Proteintech), MMP13 (Proteintech), ADAMTS-5 (Abcam), collagen II (Abcam), SOX9 (Proteintech), Aggrecan (Abcam), Bcl-2 (CST), Bax (CST), cleaved caspase-3 (CST), NLR Family Pyrin Domain Containing 3 (NLRP3, Affinity), apoptosis-associated speck-like protein containing a CARD (ASC, CST), cleaved caspase-1 (CST), gasdermin D (GSDMD, Affinity), IL-1β (CST), IL-18 (CST), phosphorylated IκBα (p-IκBα, Affinity), IκBα (Affinity), p-p65 (CST), p65 (CST), and GAPDH (CST) at 4° C. Dilute the primary antibody at 1:1000. After incubation by secondary antibody (1:1000) for 90 minutes, ECL luminescent solution exposed WB bands. We followed our previous methods [19 (link)].

Mouse decidua and human decidual tissue were extracted with lysis buffer, and the protein concentration was detected with a BCA protein quantification kit (Beyotime, Shanghai, China). The total lysate was separated by 10%-12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane. The membranes were incubated overnight with HMGB1 (1:10000 dilution; Abcam, Cambridge, UK), GSDMD (1:500 dilution; Affinity, USA), caspase-1 (1:1000 dilution; Abcam, Cambridge, UK), NLRP3 (1:200 dilution; NOVUS, USA), TLR2 (1:500 dilution; Abcam, Cambridge, UK), TLR4 (1:500 dilution; Abcam, Cambridge, UK), RAGE (1:1000 dilution; Abcam, Cambridge, UK) antibodies in a shaker at 4°C overnight, β-actin (1:1000 dilution; Abcam, Cambridge, UK) was used as an internal reference control. Membranes were incubated with secondary antibodies for 2 hours after washing the next day, and the ECL detection kit (Pierce Biotechnology, Rockford, USA) was then used for signal detection.