α sma

Proteins were isolated from the ventricular homogenate or cells with lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China) containing phenylmethylsulfonyl fluoride (PMSF) (Sigma-Aldrich) and subjected to Western blot, as described previously [24 (link)]. The antibodies used in this study were glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and collagen type III (Col3al) (Proteintech, Rosemont, IL, USA), matrix metalloproteinase 2 (MMP2) and MMP9 (Abcam, Cambridge, MA, USA), α-SMA (Bioss, Beijing, China), cellular communication network factor 2 (CCN2) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and TNF-α, p-AKT (Ser473), AKT, p-ERK (Thr202/Tyr204), ERK, p-STAT3 (Tyr705), STAT3, and Caspase-3 (Cell Signaling Technology, Danvers, MA, USA).

Proteins samples were extracted from primary HSCs and LX‐2 cells using RIPA lysis buffer (Beyotime, China). Equal amounts of proteins were separated by 10% SDS‐PAGE and transferred onto PVDF membranes (Millipore, USA). The membranes were blocked with 5% skim milk for 1 hour at room temperature and then incubated with primary antibodies overnight at 4°C. The following antibodies were used for Western blotting: β‐actin (1:500; Bioss, China), Col1α1 (1:500; Bioss), α‐SMA (1:500; Bioss), PLK1 (1:1000; Abcam), Bax (1:800; Abcam), Bcl‐2 (1:800; Abcam), cleaved caspase‐3 (1:1000; Abcam), β‐catenin (1:500, Bioss), c‐Myc (1:500; Bioss) and Cyclin D1 (1:500; Bioss). The membranes were incubated with HRP‐conjugated secondary antibodies (1:10000; ZSGB‐Bio, China) for 60 minutes at room temperature. The protein bands were detected with a chemiluminescent (ECL) system (Bio‐Rad, USA) and analysed using IMAGE J software (National Institutes of Health, USA).

Sections were block with 5% bull serum albumin (BSA) for 20 min and incubated with antibodies against TGF-β₁ (1:150 dilution, Bioss, Beijing, China), α-SMA (1:100 dilution, Bioss, Beijing, China), COL-I (1:150 dilution, Bioss, Beijing, China), COL-III (1:120 dilution, Bioss, Beijing, China), at 4°C for 12 h, followed by incubation with goat anti-rabbit immunoglobulin G (ZSGB-BIO, Beijing, China) at 25°C for 2 h, then the sections were counterstained with hematoxylin. The expressions of up-mentioned proteins were observed with a Leica microscope, and images were collected for semi-quantitative analysis achieved by Image-Pro Plus 6.0 professional image acquisition and analysis system (Media Cybernetics, Rockville, MD, United States).

For formalin‐fixed paraffin‐embedded tissues, slides were de‐waxed by washing with xylenes, rehydrated and blocked. For cultured cells, cells were seeded in chamber slides (Cat. #: C6807; Sigma Chemical Co.), fixed with 4% formaldehyde, permeabilized in 0.1% Triton‐X100, and blocked. Slides were then incubated with primary antibodies for α‐SMA (Cat. #: bs‐0189R; Bioss, Beijing, China), Vimentin (Cat. #: 5741; Cell Signaling Technology, Danvers, MA, USA), E‐cadherin (E‐cad, Cat. #: 14472; Cell Signaling Technology), or SP‐C (Cat. #: sc‐7706; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), for overnight at 4°C. Slides were then washed and stained with fluorophore‐conjugated secondary antibodies for 1 hr at room temperature. Cover slips were mounted using mounting medium (Cat. #: C9368; Sigma Chemical Co.), allowed to harden overnight at 4°C and then sealed. Images were acquired on Olympus IX70 (Japan) fluorescence microscope.

ASIC1a (1:1,000 dilution, Bioss), Collagen-1 (1:1,000 dilution, Proteintech), α-SMA (1:1,000 dilution, Bioss), β-Actin (1:1,000 dilution, Proteintech), CaM (1:1,000 dilution, Affinity), CaMK II (1:1,000 dilution, Affinity), NFAT (1:1,000 dilution, Affinity), MMP-13 (1:1,000 dilution, Bimake.cn), NF-κB (1:1,000 dilution, Bimake.cn), KN93, and colchicine were purchased from MedChemExpress (MCE, United States). Spider-venompeptide (PcTx-1) was purchased from Abcam (Cambridge, United States). High-sugar DMEM culture medium and phosphate buffered saline (PBS) were purchased from HyClone (United States). Horseradish enzyme labeled anti-rabbit IgG and horseradish enzyme labeled anti-mouse IgG were purchased from Bioss (Beijing, China). PVDF membrane was purchased from Millipore (United States). Fluo-3AM calcium fluorescent probe was purchased from beyotime (Shanghai, China).Lipofectamine 2000, TRIzol Reagent, and Opti-MEM were purchased from Invitrogen (Invitrogen, United States). Amiloride was purchased from Sigma-Aldrich (St. Louis, MO, United States). Rapid Gel Preparation Kits were purchased from EpiZyme (EpiZyme, China). Cell cycle detection kit and CCK-8 cell proliferation toxicity assay kit were purchased from BestBio (Beijing, China).

After 21 days of induction, the 2D and 3D cultured samples were rinsed with PBS and subjected to immunofluorescence stainings. After fixing with ice-cold methanol for 5 min, the sections were permeabilized with 0.1% Triton-X100 for 5 min, blocked with 1% BSA for 10 min, and then incubated with rabbit polyclonal anti-vWF antibody (1 : 100, Abcam, UK), rabbit polyclonal anti-VEGFR2 antibody (1 : 100, Novus, USA), and rabbit monoclonal anti-CD31 antibody (1 : 100, Novus, USA) for 2 h at 37°C. After 3 washes in PBS, Texas Red goat anti-rabbit IgG (1 : 300, Molecular Probes, USA) was added for 1 h. Complexes were incubated with DAPI for nuclei staining. Noninduced 2D and 3D hADSCs incubated in completed medium (DMEM supplemented with 10% FBS, 100 U/mL penicillin, and 100 g/mL streptomycin) served as the negative controls while HUVEC served as the positive control. Besides, the immunofluorescence staining of α-SMA (1 : 100, Bioss, China) was examined in these 4 groups after 7 days in vitro. All of the samples were examined under optical microscopy.

Total proteins were extracted from liver tissue and cells using radio-immunoprecipitation buffer (RIPA). 80 μg of sample proteins were separated by in 10 or 12% SDS-PAGE gel, and transferred onto PVDF membranes (Millipore Corporation, Billerica, MA, USA) by electrobloting. The membranes were blocked for 60 min in a buffer containing 0.1% Tween-20 and 5% milk. The membranes were incubated overnight at 4 °C with primary antibodies against α-SMA, Col-1, MMP-2 (Bioss, Beijing, China), Smad7 (Novus Biologicals, Littleton, USA), Wnt1 (Abcam, Cambridge, MA, USA), Wnt5a (Novus Biologicals, Littleton, USA), β-catenin, GSK-3β (ProteinTech Group, Chicago, USA), NFAT5 (Santa Cruz, CA, USA). Immune complexes were detected with horseradish peroxidase (HRP)-conjugated secondary antibodies (ProteinTech Group, Chicago, USA), and then were visualized by ECL method. β-actin (Boster, Wuhan, China) was served as a loading control. Intensity of each protein band of interest was quantified by densitometry using Quantity One 4.6.3 software (Bio Rad).