Timp1

Total proteins were extracted from liver tissues and cells using radio-immunoprecipitation buffer (RIPA). Equal amounts of denatured proteins were separated on 10% SDS-PAGE and analyzed by immunoblotting with primary antibodies against GCLC (1:1000; Proteintech, 12601-1-AP), α-SMA (1:1000; Proteintech, 14395-1-AP), COL1 (1:1000; Abcam, ab6308), glucose-regulated protein 78 (GRP78; 1:1000; Proteintech, 11587-1-AP), CCAAT/enhancer-binding protein homologous protein (CHOP; 1:1000; Proteintech, 15204-1-AP), phosphorylated inositol-requiring enzyme 1 (p-IRE1; 1:1000; Abcam, ab48187), endoplasmic reticulum degradation enhancer, mannosidase alpha-like 1 (EDEM1; 1:500; Proteintech, 26226-1-AP); protein kinase inhibitor (p58^IPK; 1:1000; Abcam, ab70840); nuclear factor kappa B (NF-κB; 1:1000; Proteintech, 14220-1-AP), inhibitor of KB kinase b (IKKB; 1:1000; Proteintech, 15649-1-AP), tumor necrosis factor α (TNFα; 1:1000; Abcam, ab6671), transforming growth factor 1 (TGFβ1; 1:1000; Proteintech, 21898-1-AP), tissue inhibitor of the metalloproteinase (TIMP1; 1:1000; Proteintech, 16644-1-AP), Matrix metalloproteinase 2 (MMP2; 1:1000; Proteintech, 10373-2-AP), and β-actin (1:10000; Proteintech, 14395-1-AP). After the incubation with HRP-conjugated secondary antibodies, the signals were visualized by the ECL system (Thermo Fisher Scientific).

After the MDA-MB-231 cells were treated with XLLXF (50, 100, and 200 µg/mL) for 24 and 48 h, total cell protein lysates were extracted using RIPA lysis buffer that contained protease and phosphatase inhibitor cocktails. Protein lysates (20 µg), which were determined by BCA analysis (Beyotime, China), were loaded onto 10% SDS-PAGE gels. The protein bands were transferred onto NC membranes and blocked with 5% non-fat milk for 1 h at room temperature. The NC membranes with proteins were incubated with diluted primary antibodies at 4 °C overnight. The primary antibodies used in the analyses were as follows: VEGFA (1:1,000, Proteintech), MMP2 (1:1000, Proteintech), MMP9 (1:1000, Cell Signaling Technology), Vimentin (1:1000, Proteintech), VE-cadherin (1:1,000, Cell Signaling Technology), TIMP-1 (1:1000, Proteintech), TIMP-3 (1:1,000, Proteintech), and Twist1 (1:1000, Proteintech). The membranes were incubated with relative sources of secondary antibodies (1:5000) at room temperature for 1 h. Specific protein bands were recognized with Immobilon Western Chemiluminescent HRP Substrate (Millipore, MA, USA). Image J software was used for image analysis.

Total protein was extracted from lung tissues or cells using Tissue or Cell Total Protein Extraction Kit (Sangon Biotech). Equivalent protein from different samples was separated by protein electrophoresis, following by transformation onto PVDF membranes (Merck Millipore, Billerica, MA, USA). The membranes were incubated with the anti-rabbit SIRT1, TIMP-1, MMP-9, CHOP, GRP78, caspase-12 or caspase-3 antibodies (1:1000 dilution, Proteintech, Wuhan, China) at 4 °C overnight after immersed into blocking buffer. After the membranes were washed with TBST for several times, goat anti-mouse IgG antibody (1:5000, Proteintech) labelled with horseradish peroxidase were incubated with the membranes as a secondary antibody. Anti-mouse β-actin antibody (1:5000, Proteintech) was used as a reference protein for normalization. The grey levels of the protein bands were examined by Image J software.

Propylene glycol alginate sodium sulphate was purchased from Dalian Tianyu Pharmaceuticals Co., Ltd and disposed in saline to obtain different drug doses of 12.5, 25 and 50 mg/kg, and stored at 4°C.¹² CCl₄ was acquired from Sinopharm. Microplate test kits of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were obtained from Nanjing Jiancheng Bioengineering Institute (Jiancheng Biotech). Antibodies against IL‐6, Col‐1, α‐SMA, TGF‐β1, MMP‐2, TIMP‐1, Beclin‐1, p62, Smad2 and Smad3 were purchased from Proteintech, and those against p‐Smad2, p‐Smad3, JAK2, STAT3 and p‐STAT3 were purchased from Cell Signaling Technologies. The polymerase chain reaction (PCR) kit was acquired from Takara Biotechnology.

Cells at the logarithmic phase were harvested and lysed with radioimmunoprecipitation assay (RIPA) buffer [150 mM NaCl, 1% NP-40, 0.25% Na-deoxycholate, 1 mM ethylenediaminetetraacetic acid (EDTA), 50 mM Tris-HCl, pH 7.4]. Total protein and nuclear proteins were extracted. Proteins were separated by 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membranes were incubated with a primary antibody overnight at 4 °C, followed by a secondary antibody (Zhongshan Bio-Tech Co, Beijing, China) for 1 h at room temperature. Then, the membranes were scanned using an Odyssey infrared imaging system (LICOR, Lincoln, NE, USA). The primary antibodies against Mmp13, Timp1, Bsp, Runx2 (Proteintech Group, Inc., Wuhan, China), and GADPH (KangChen Biotech, Shanghai, China) were used for western blot analysis.