3.9. The Antifibrotic Effects on the Human HSC Line LX-2 Cells

After ultraviolet sterilization, raw IMB16-4, IMB16-4-MSNs and MSNs were suspended in water, respectively. Another group of raw IMB16-4 was suspended in DMSO. The concentration of IMB16-4 was 2 mM. Then, 2µL of solution containing IMB16-4 was added into 2 mL of serum-free culture with TGFβ1. LX-2 cells were cultured as described above. LX-2 cells were seeded in a 6-well plate, cultured in DMEM/GlutaMAX I, containing 10% fetal bovine serum (FBS) in 5% CO₂ atmosphere at 37 °C. Serum-free culture was replaced until the cells reached 90 ≈ 95% confluence. After 24 h, cells were treated with TGFβ1 (2 ng/mL) and IMB16-4 (2 µM) for 24 h. Then, cells were washed with phosphate buffer saline (PBS) and protein was extracted in radio-immunoprecipitation assay (RIPA) buffer. Then mixture was centrifuged at 12,000 rpm at 4 °C for 20 min. The supernatant was collected, and the total protein was determined using the bicinchoninic acid (BCA) protein assay kit (Beyotime Biotechnology, China). Samples were applied to the 10% SDS-PAGE gel. Then, the protein bands were transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore Corp, Atlanta, GA, USA) and the membrane was blocked for 1 h with 5% nonfat milk. The membrane was then incubated with desired primary antibodies overnight at 4 °C followed by horseradish peroxidase (HRP) conjugated secondary antibodies (1:10,000) at room temperature for 1 h. The protein bands were analyzed on an imager (Tanon5200, China).