α sma

Total liver protein was isolated using RIPA lysis buffer (CWBIO, Beijing, China) containing protease inhibitor cocktail (CWBIO). Equal amounts of protein were separated using 8% SDS‐PAGE gel electrophoresis in a Bio‐Rad Mini‐Protean system. The separated proteins were then transferred to nitrocellulose membrane (Millipore, MA) and incubated with specific primary antibodies (aSMA, ID1, HGF, WNT2, c‐MET, GSK‐3β, Proteintech, Wuhan, China; phosphor‐c‐MET, phosphor‐GSK‐3β, Abclonal, Wuhan, China). After washing and incubation with the secondary antibody, antibody complexes bound to specific liver proteins were visualized using the BIO‐RAD Gel Doc XR+ system (Bio‐Rad, CA).

Immunofluorescence staining was analyzed using an Olympus microscope. The expression and distribution of PRDX2 (1:100; Abcam, USA and Proteintech, China), SM22α (1:100; Proteintech, China), and α-smooth muscle actin (α-SMA, 1:200; Proteintech, China) were detected. Moreover, the osteopontin (OPN, 1:100; Abcam, USA) was evaluated to mark the phenotype switching of the CAVSMCs. The nuclei were stained with 0.5 μg/ml 4′,6-diamidino-2-phenylindole (DAPI; Solarbio, China).

After measurements were taken, the heart was quickly removed and washed with PBS. The atrial tissue was isolated and fixed with 4% paraformaldehyde, embedded in paraffin and sectioned into 5 microns slices. The cultured fibroblasts were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.2% Triton X-100 for 20 min, and finally blocked with 3% BSA for 60 min at room temperature.
Atrial fibrosis was quantified by analysis of images of Masson's trichrome-stained samples. The atrial samples were incubated with primary antibodies against SK4 (Abcam, USA), α-SMA (Proteintech Group Inc., China), and DDR-2 (Santa Cruz Biotechnology, USA) and subsequently with secondary antibodies FITC-conjugated AffiniPure goat anti-rat IgG and CY3-conjugated AffiniPure goat anti-rabbit IgG (ASPEN Biotechnology, China). Fibroblasts were incubated with primary antibodies against SK4 and α-SMA and subsequently with secondary antibodies CY3-conjugated AffiniPure goat anti-rabbit IgG and FITC-conjugated AffiniPure goat anti-rat IgG (ASPEN Biotechnology, China). The nucleus was stained with DAPI. The results were analyzed by Image-Pro Plus 6.0 software (Media Cybernetics, USA). Three visual fields in each sample were assessed randomly at 400× magnification.

HDAC6 (Cell Signaling Technology Cat# 7612, RRID:AB_10889735; 1:1000 in WB, 1:100 in IHC), NLRP3 (Cell Signaling Technology Cat# 13158, RRID:AB_2798134; 1:1000 in WB), NLRP3 (AdipoGen Cat# AG-20B-0014, RRID:AB_2490202; 1:1000 in WB), Caspase-1 (Proteintech Cat# 22915-1-AP, RRID:AB_2876874; 1:3000 in WB), Caspase-1 (AdipoGen Cat# AG-20B-0042, RRID:AB_2490248; 1: 1000 in WB), Cleaved N-terminal GSDMD (Abcam Cat# ab215203, RRID not available; 1:500 in WB), ASC (Santa Cruz Biotechnology Cat# sc-514414, RRID:AB_2737351; 1:500 in WB), α-Tubulin (Proteintech Cat# 11224-1-AP, RRID:AB_2210206; 1:5000 in WB), β-Tubulin (Proteintech Cat# 10068-1-AP, RRID:AB_2303998; 1:5000 in WB), acetylated α-Tubulin (Lys40) (Proteintech Cat# 66200-1-Ig, RRID:AB_2722562; 1:1000 in WB), α-SMA (Proteintech Cat# 14395-1-AP, RRID:AB_2223009; 1:2000 in WB, 1:300 in IHC), Col1a (Cell Signaling Technology Cat# 72026, RRID:AB_2904565; 1:2000 in WB, 1:300 in IHC), Goat anti-mouse IgG (H + L), HRP conjugate (SA00001-1, Proteintech), Goat anti-rabbit IgG (H + L), HRP conjugate (SA00001-2, Proteintech), Donkey anti-rabbit IgG (H + L), Alexa Fluor 488 (A-21206, ThermoFisher), Donkey anti-mouse IgG (H + L), Alexa Fluor Plus 555 (A32773, ThermoFisher).

Protein samples were separated by 8%, 10%, or 12% sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) and then transferred onto PVDF membranes (EMD Millipore, Burlington, MA, USA). After being blocked with 5% non-fat milk, the membranes were incubated with the following primary antibodies at 4°C overnight: α-tubulin (GeneTex, Irvine, CA, USA), collagen I (GeneTex), α-SMA (Proteintech, Chicago, IL, USA), TGF-β (Cell signaling technology, Danvers, MA, USA), TNF-α (GeneTex), NLRP3 (Proteintech), caspase-1 (Proteintech), IL-18 (Proteintech), IL-1β (Proteintech), caspase-3 (GeneTex), caspase-8 (GeneTex), caspase-9 (GeneTex), and BCL-2 (GeneTex). Membranes were then incubated with HRP-conjugated mouse anti-IgG (EMD Millipore) or HRP-conjugated rabbit anti-IgG (EMD Millipore) secondary antibodies for 1 h. Membranes were developed using ECL detection reagent (EMD Millipore). Relative protein levels were quantified using Image J (Version 1.46, National Institute of Health, Bethesda, MD, USA), and protein densitometry were expressed relative to that of α-tubulin.