Mmp 9

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).

(1) After the cells in each group were washed with PBS for 3 times, RIPA lysate (Meilunbio, Dalian, China) was used to isolate total protein from HA-VSMCs, and extracted proteins were quantified by a BCA Protein Quantification Kit (Epizyme, Shanghai, China). (2) SDS-PAGE gel (Epizyme, Shanghai, China) electrophoresis was performed, and then the proteins were transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, USA) and blocked with 5% slim milk for 1 h. (3) The membranes were incubated overnight at 4 °C together with relevant primary antibodies and then incubated with corresponding secondary antibody for 2 h at room temperature. (4) The protein bands were visualized by enhanced chemiluminescence (Vazyme, Nanjing, China) and quantified by Image Lab (LI-COR). β-actin, PCNA and MMP-9 antibodies were purchased from Absin (Beijing, China), while Notch1, Jagged-1, ERK1/2 and p-ERK1/2 were from Abmart (Shanghai, China).

The expression of Caspase-3, Caspase-8, Bcl-2, Bax, MMP2, MMP9, VEGF, Occludin, Claudin and STAT3 from SPG-56 treated mice tumors, and the expression of MMP2, MMP9, VEGF, Occludin, Claudin and STAT3 from control livers, were analyzed by western blotting. The procedure for immunoblotting followed a previous report²¹ . Total protein was extracted from the cells by 1X RIPA buffer (BBI life sciences, China) containing 1 mM phosphatase inhibitor cocktail and 1 mM phenylmethylsulfonyl fluoride (PMSF). Equalamounts of proteins (10 μg) were subjected to SDS-PAGE. After transferred onto a polyvinylidene fluoride membrane, they were incubated with primary antibodies against β-actin, Caspase-3, Caspase-8, Bcl-2, Bax, VEGF, Occludin, Claudin, STAT3(Proteintech Group, Inc., USA), MMP2 and MMP9(Absin Bioscience Inc., China), respectively (1:1000 dilution, 4 °C, overnight). Western blot was quantified by using ImageJ software.