D3r6y

A time course of immunostaining of Caspase-3 and NF-κB was carried out on DBA/2J mice. The time point selected was 4 weeks. After dewaxing, the cochlear tissues were subjected to heat induced antigen retrieval in citrate buffer (0.01 mol/L, pH 6.0), washed three times in 1 × PBS at room temperature for 5 min, and permeabilized with 3% H₂O₂ for 10 min. The slides were then blocked with 3% bovine serum albumin (BSA) and 3% goat serum at 37^°C for 1 h and stained with rabbit anti-cleaved Caspase-3 antibody (1:250 dilution) (D3R6Y, Cell Signaling Technology Inc., Danvers, MA, United States) or NF-κB antibody (1:50 dilution) (P65, Santa Cruz Biotechnology Inc., Santa Cruz, CA, United States) at 4^°C overnight. Following a thorough wash with 1 × PBS for 5 min, the sections were immersed in Goat anti-rabbit IgG(H + L) HRP (1: 400 dilution) (Sparkjade, China)and Goat anti-mouse IgG(H + L) HRP (1: 400 dilution) (Absin, China) at 37^°C for 30 min, and then counterstained with 4 Detection Kit (ZLI-9018 DAB kit, ZSGB-BIO, China) for 15 min and with Harris hematoxylin (Sigma, China) for 1 min at room temperature. The slides were sealed using neutral gum and examined under a scanning confocal microscope.

Cochlear tissue was ground with Radioimmunoprecipitation assay (RIPA) buffer (Beyotime, Jiangsu, China) containing phosphatase inhibitors (Roche, Basel, Switzerland) and phenylmethylsulfonyl fluoride (PMSF) (Beyotime). Total protein samples were separated by electrophoresis in 15% SDS polyacrylamide gels (Servicebio, China) and electrotransferred to a nitrocellulose membrane (Millipore, Billerica, MA, United States). The membrane was subsequently blocked with 5% non-fat milk for 1 h. Afterward, the membrane was incubated at 4°C overnight with the following primary antibodies: anti-NF-κB (1:2000 dilution) (P65, Santa Cruz Biotechnology Inc., Santa Cruz, CA, United States), anti-Caspase-3 (1:1000 dilution) (D3R6Y, Cell Signaling Technology Inc., Danvers, MA, United States).
Next day, the membrane was washed three times with Tris-buffered saline-Tween 20 (TBST) buffer, incubated with corresponding horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature and then washed with TBST buffer three times. The immunoreactive proteins on the blots were visualized using the ECL reagent (Sparkjade Biotechnology, China) and detected using a chemiluminescence imaging system (ChemiScope 6200 Touch; Clinx Science Instruments Co., Ltd., Shanghai, China). The protein bands were quantified by detecting the gray values using ImageJ software.

Standard techniques were used for protein quantification, separation, transfer, and blotting in KLE and HEC-1A cells. Primary antibodies immunoblotting antibodies were used: ERRα (1: 500, ab76228, Abcam, London, UK), ERRα (1:500, E1G1J, Cell Signaling Technology, Massachusetts, USA), NLRP3 (1:500; 19,771-1AP, Proteintech, Wuhan, China), HIF-1α (1:500; 20,960-1AP, Proteintech, Wuhan, China), cleaved GSDMD (1:500; #36,425, Cell Signaling Technology, Massachusetts, USA), GSDMD (1:500, E9S1X, Cell Signaling Technology, Massachusetts, USA), GSDME (1:500, 84005S, Cell Signaling Technology, Massachusetts, USA), Caspase1 (22,915–1-AP, Proteintech, Wuhan, China), Caspase-1 (YT5743, Immunoway, USA), Caspase-1 (1:500, D7F10, Cell Signaling Technology, Massachusetts, USA), Caspase-3 (1:500, D3R6Y, Cell Signaling Technology, Massachusetts, USA), IL-18 (1:500,10,663–1-AP, Proteintech, Wuhan, China), cleaved-IL-1β (1:500, D3A3Z, Cell Signaling Technology, Massachusetts, USA), β-actin (1:2000, YM3028l, Immunoway, USA), β-tublin (1:2000, YM3030, Immunoway, USA), and GAPDH (1:2000; YM3029, Immunoway, USA).