Gapdh

A Xuesaitong (XST) dispersible tablet was obtained from Yige Pharmaceutical Co. Ltd. (Xiangtan, Hunan, China). Streptozotocin (STZ) was obtained from Sigma-Aldrich company (Sigma-Aldrich, USA). Antibodies of PTEN, PDK1, Nox4, nephrin, and 4′,6-diamidino-2-phenylindole (DAPI) were purchased from Abcam (Cambridge, UK); antibodies of Akt, p-Akt, mTOR, and p-mTOR were acquired from Cell Signaling Technology (Danvers, MA, USA). WT1, GAPDH, and β-Tubulin were purchased from Absin Bioscience Inc. (Shanghai, China). Antibodies of horseradish peroxidase-conjugated rabbit and mouse IgG were obtained from Beyotime Institute of Biotechnology (Shanghai, China). The assay kit for terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) was obtained from Shanghai Yisheng Chemical Company Limited (Shanghai, China). The assay kit for bicinchoninic acid (BCA) protein was obtained from Biosharp Biotechnology Company Limited.

PDLSCs were washed with cold phosphate buffer solution (PBS) and then incubated in RIPA buffer containing proteinase inhibitor and phosphatase inhibitor (Beyotime) on ice. After ultrasonic lysis and centrifugation, protein solutions were collected, and the concentrations of protein were detected by a BCA assay kit (Biotime). All protein samples were separated by SDS-PAGE gels and transferred to PVDF membranes (Millipore, USA). After being blocked with 5% nonfat-dried milk solution (Beyotime) for 1 hour, the membrane was incubated with primary antibodies overnight at 4 °C, including Erk (#4695, CST, USA), phosphorylated-Erk (p-Erk, #9101, CST), RUNX2 (#8486, CST), COL1 (#39952, CST), EID3 (ab124447, Abcam, UK), GAPDH (abs132004, Absin Bioscience, China), and β-actin (BM3873, Boster, China). Then, the membrane was incubated with secondary antibodies for 1 hour at room temperature. Finally, the protein bands on the membrane were detected by enhanced chemiluminescence (Millipore, Billerica, MA, USA) under an Amersham Imager 600.

Recombinant human/mouse/rat activin A was provided by R&D systems (Minneapolis, MN, USA). Recombinant murine TNF-α and recombinant murine SDF-1α (CXCL12) were obtained by PeproTech (Rocky Hill, NJ, USA). Cell Counting Kit-8 (CCK-8) was bought from GlpBio Biotechnology Co. (Shanghai, China). NO detection kit was purchased Beyotime Biotechnology Co. (Shanghai, China). Enzyme-linked immunosorbent assay (ELISA) kit for TGF-β1 was obtained from eBioscience (San Diego, USA); ELISA kit for IL-6 was provided by R&D systems (Minneapolis, MN, USA). Reverse transcription-PCR (RT-PCR) kit was purchased from Takara Biotechnology Co. (Dalian, China). The antibodies used for Western blotting were as follow: ActRIIA (Absin), Smad3 (Immunoway), p-Smad3 (Abcam), ERK1/2 and p-ERK1/2 (Cell Signaling), MMP-2 (Absin), MMP-9 (Absin) and GAPDH (Absin).

A quantity of 10 μg of protein was loaded onto a 10% gradient polyacrylamide gel, electrophoretically transferred to a polyvinylidene difluoride membrane and probed with the following primary antibodies: S100A8/A9 (1:1000, Abcam, UK), Col1 (1:2000, Proteintech, China), α-SMA (1:6000, Proteintech, China), JAK2 (1:1000, Proteintech, China), p-JAK2 (Tyr1007) (1:1000, Absin, China), STAT3 (1:2000, Proteintech, China), p-STAT3(Tyr705) (1:1000, Absin, China), RAGE(1:1000, Proteintech, China), TLR4 (1:1000, Proteintech, China),  and GAPDH (1:1000, Absin, China), which was used as an internal control. Secondary antibodies were horseradish peroxidase-conjugated to mouse anti-rabbit/mouse IgG (1:5000, Proteintech, China).

To collect and lyse HCC cells, radioimmunoprecipitation assay buffer (RIPA, Solarbio, China) was used. To decrease protease activity, phenylmethylsulfonyl fluoride (Solarbio, China) was added throughout the lysis process. After measuring total cell protein concentrations using a NanoDrop One ultraviolet spectrophotometer (Thermo Scientific), a protein loading buffer (Beyotime Institute of Biotechnology, China) was added, and samples were heated to denature proteins. After that, the protein samples were isolated using a 10% PAGE Gel Fast Preparation Kit (EpiZyme, China) and transferred on 0.45-μm polyvinylidene fluoride membranes (PVDF, Millipore, Germany). The PVDF membranes were then blocked at room temperature for 120 min. PVDF membranes were treated with primary antibodies overnight at 4 °C after being diluted at 1:1000. Then, membranes were incubated with appropriate secondary antibodies (Abcam, USA) for 1 h, and the bands were detected using enhanced chemiluminescence reagents (New Cell & Molecular Biotech, China) and quantified using ImageJ software (NIH Image, Bethesda, MD). The following primary antibodies were used in this study: MCUR1 (Cell Signaling Technology, USA), GAPDH (Absin, China), and P-gp, GP IIb and GP VI (Abcam, UK).