Sa00013 2

Immunocytochemical staining was performed on the coverslips obtained from each of the experimental groups, with the use of rabbit polyclonal antibodies against pERK (1:300, WLP1512, Wanleibio, Shenyang, China). Briefly, the coverslips were washed three times with PBS, permeabilized with 0.3% Triton X-100 for 15 min at room temperature, incubated with 3% H₂O₂ for 10 min, blocked by normal goat serum for 30 min at 37 °C, and then with the appropriately diluted first antibody at 4 °C overnight in a humid chamber. The color reaction was carried out using 3,30-diaminobenzidine tetrahydrochloride (DAB). For immunofluorescence labeling, the THJ-16T or THJ-21T-bearing coverslips of the experimental groups were rinsed three times with PBS, permeabilized with 0.3% Triton X-100 for 15 min, blocked with 10% goat serum in PBS for 30 min at 37 °C, then incubated with primary antibody pSTAT3 (1:300, ab76315, abcam, Cambridge, UK) overnight at 4 °C, followed by Coralite488-conjugated goat anti-rabbit IgG (1:500, SA00013-2, Proteintech, Wuhan, China) at 37 °C for 60 min in the dark. Nuclei were labeled with Hoechst 33342 (C1025, Beyotime Biotech, Shanghai, China), and images captured by fluorescence microscope (Nikon, ECLIPSE Ni-U).

The tissues were cut into 5 μm cross sections, then stained with hematoxylin and eosin (HE) to observe the morphological changes and Masson’s trichrome stain to evaluate interstitial fibrosis. Image-Pro Plus 6.0 software (Media Cybernetics Co., Ltd., Carpenteria, CA, USA) was used to examine cell morphology and observe the volume percentage of ventricular interstitial collagen.
Harvested hearts were fixed and embedded in optimal cutting temperature compound (OCT) (VWR, PA) for immunofluorescence staining. Then, the hearts were cut into 5 μm cross sections and stained with primary antibody for Ac-FOXO3a (Lys271) (1:200; AF3771, Affinity) overnight at 4 °C. Alex 488-conjugated goat anti-rabbit antibody (1:200, SA00013-2, Proteintech) was used as a secondary antibody. DAPI was used to stain the nuclei. Images were acquired under an Olympus inverted microscope (IX81, Tokyo, Japan). ImageJ was used to calculate the intensity of the fluorescent light.

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).

All collected sections were dewaxed with dimethylbenzene, soaked in graded ethyl alcohol, and washed with distilled water. Parts of the sections were stained with hematoxylin and eosin (H&E), dehydrated with gradient alcohol (95%–100%), and sealed. Urea and trypsin antigen were used to repair other parts of the sections, which were subsequently treated with primary and secondary antibodies, and then DBA was added for 5 min to develop color and observed with a microscope. Other sections were immersed in EDTA buffer (pH 9.0), heated, and then cooled to room temperature. Before adding the primary and secondary antibodies, the sample was first cleaned with 0.01M PBS (pH 7.4–7.6), submerged in 75% alcohol, Sudan black dye solution was added, and then stained with DAPI working solution at 37°C until sealed. The H&E staining solution and immunohistochemistry reagents and kits were purchased from Wellbio (K435960 and K484350) and Beijing ZSGB company (600D54 and 600W23), respectively. The immunofluorescence reagents were purchased from ProteinTech (SA00013-2). The stained cartilages were observed and imaged using a light microscope (BA410T, Motic China Group Co., Ltd., Xiamen, China). Immunohistochemical sections were examined for IL-1β, IL-6, TNF-α, and IFN-γ. Immunofluorescence sections were used to observe the expression of NF-κB p65.

After dewaxing, rehydration and antigen-repair, the slides were incubated with Triton-X-100 for 10 min at room temperature and blocked with goat serum (ZSGB-BIO) for 30 min at 37 °C. The sections were incubated with the primary antibody rabbit anti-G-protein coupled receptor 30 (1:50; GTX107748, GeneTex, USA), rabbit anti-Phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (1:200; #4370, Cell Signaling Technology) or rabbit anti-ZO-1 (1:50; 21773-1-AP, Proteintech) at 4°C overnight. The slides were incubated with Rhodamine (TRITC)-conjugated Goat Anti-Rabbit IgG (H + L) (1:50; SA00007-2, Proteintech) or CoraLite488 conjugated Goat Anti-Rabbit IgG (H + L) (1:50; SA00013-2, Proteintech) as secondary antibody in a humid dark box at 37 °C for 60 min. 4’6-diamidino-2-phenylindole (DAPI) was used to label the nuclei. After the preparation against quenching, the slides were observed under fluorescence microscope (Nikon, Japan).

After using 4% paraformaldehyde immobilization at room temperature, 0.1 Triton X-100 permeation at room temperature, and 10% goat serum block at 37°C for 30 min, respectively, Piezo1 antibody (15939-1-AP, Proteintech, 1:200) was used to incubate overnight at 4°C. Then CoraLite488-conjugated secondary antibody (SA00013-2, Proteintech, 1:300) was incubated for 1 hour and DAPI (Biosharp) for 20 min at 37°C. Pictures were taken under a fluorescence microscope (Olympus, Japan) with the same exposure time. Moreover, the individual cell was randomly grabbed in the images by image processing software Image-J (Version 1.52 V, National Institutes of Health, USA) to analyze the fluorescence intensity.

Myocardial tissue was cryo-embedded by optical coherence tomography, and then, 10-μm cryo-sections were generated. Cryo-sections and glass bottom plates (cultured endothelial cells) were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and blocked with 2% bovine serum albumin and incubated with different primary antibodies at 4 °C overnight according to the corresponding experiments, including Anti-Cardiac Troponin I, CD31, VEGF-A, α-SMA, wheat germ agglutinin (WGA) (L4895, Sigma-Aldrich, 1: 100 dilution), and KMT2D. The next day, Alexa-488-(SA00013-2, Proteintech, 1: 1000) or Alexa-568-conjugated secondary antibodies (A11031, Invitrogen, 1: 1000) were incubated for 1 h, and finally, the nuclei were stained with DAPI (D9542, Sigma). All sections and glass bottom plates were examined by confocal microscope (ECLIPSE Ti A1, Nikon).