Yt5743

The cells were fixed with 4% paraformaldehyde (PFA, Solarbio, China) for 15 min, washed with PBS, permeabilized with 0.5% Triton X-100 for 30 min, and blocked with 5% goat serum (Solarbio, China) for 1 h at room temperature. Then, the cells were incubated with the primary antibodies in blocking buffer overnight at 4°C. After washing three times with PBS, the cells were incubated with the fluorescence-labeled secondary antibodies Alexa Fluor® 555 (1:500, ab150078, abcam), Alexa Fluor® 488 (1:500, ab150113, abcam) or CoraLite® 488 (1:500, SA00013-2, Proteintech) for 2 h at room temperature in the dark. A staining solution consisting of 4',6-diamidino-2-phenylindole (DAPI, Solarbio, China) was added to stain the nuclei, and the images were collected under a fluorescence microscope (Olympus, Japan). For cell immunofluorescence staining counts, the number of samples per group was n=5, and 5 fields of view were randomly captured for each sample. The primary antibodies used in this study were as follows: Nestin (1:200, PA5-118114, Invitrogen), GAP-43 (1:500, ab16053, abcam), Sox-2 (1:300, 11064-1-AP, Proteintech), GFAP (1:500, ab10062, abcam), βIII-tubulin (1:500, 66375-1-Ig, Proteintech), Iba-1 (1:500, 019-19741, Wako), NLRP3 (1:200, YT5382, Immunoway), and Caspase-1 (1:300, YT5743, Immunoway).

Frozen sections of rat brain tissue with a thickness of 10 μm were harvested from each group and washed three times with PBS, treated with 0.5% Triton X 100 for 20 min, and blocked with 5% goat serum at room temperature for 1 h. The sections were incubated with the following primary antibodies overnight at 4°C: NeuN (1:200, ab104225, abcam), GFAP (1:300, ab7260, abcam), Iba-1 (1:500, 019-19741, Wako), NLRP3 (1:200, YT5382, Immunoway), and Caspase-1 (1:300, YT5743, Immunoway). The following day, the sections were incubated with the fluorescence-labeled secondary antibodies Alexa Fluor® 555 or Alexa Fluor® 488 for 2 h at room temperature in the dark. DAPI staining solution was added to the sections and observed under a fluorescence microscope (Olympus, Japan), and images were acquired. For analysis of the staining results, five samples from each group were selected, three sections were selected from each sample, five areas around the hematoma were randomly photographed in each section, and the number of positive cells was counted.

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).