Phscs

HCC cell lines MHCC97H and MHCC97H with integrin-β1 knockdown (Liver Cancer Institute of Fudan University, Shanghai, China), Hep3B and HepG2 (ATCC, USA), Huh7 (Japanese Cancer Research Bank) were grown in DMEM with 10% FBS (Gibco) and 1% penicillin/streptomycin. Cell lines were authenticated by short tandem repeat validation analysis during the study period. Primary human hepatic stellate cells (pHSCs) (Sciencell, USA) were maintained in the provided medium and LX2 cells (a gift from S. Friedman) were cultured in DMEM with 2% FBS. All cell cultures were carried out in a 37 °C incubator with a humidified atmosphere in presence of 5% CO₂.
As in our previous description [26 (link)], conditioned medium was collected from activated HSCs (HSC-CM) and anti-human POSTN antibody (2.5 μg/mL) (Abcam, Cambridge, UK) was added into HSC-CM to neutralize the activity of POSTN.
To obtain conditioned medium from calcipotriol-treated HSCs, pHSCs or LX2 cells were pre-stimulated using 10 ng/mL TGF-β1 and then incubated with 100 nM calcipotriol (Sigma-Aldrich) for 12 h, replenished with fresh medium for another 24 h and the medium was collected for the subsequent experiments.

Primary human HSCs (pHSCs; ScienCell) were grown in SteCM medium (ScienCell) in T-75 flasks coated with poly-l-lysine. When confluent, cells were trypsinized (0.05% trypsin/0.53 mm EDTA) and seeded at a ratio of 1 : 3. Subsequent passages were performed every 6 days. Immortalized human HSCs (LX-2) were kindly provided by Professor Scott L. Friedman (Mount Sinai School of Medicine). LX-2 cells were grown in high glucose DMEM containing 10% fetal bovine serum in T-75 flasks. When confluent, LX-2 cells were passaged as for pHSCs. Primary fresh human hepatocytes were purchased from 3H Biomedical. Human hepatoma (HEPG2) cells (ATCC) were grown in high glucose DMEM containing 10% fetal bovine serum in T-75 flasks according to the manufacturer's instructions. Colon carcinoma (CACO-2) cells (ATCC) were grown in MEM with Earl's salts according to the manufacturer's instructions. Cells were seeded in 24-well plates with cover slips (for Oil Red O staining, see Supplementary Material) or in 6-well plates (for immunoblot and mRNA expression analyses, see Supplementary Material).

HCC cell lines, MHCC97H (established by Liver Cancer Institute, Fudan University, Shanghai, China), MHCC97H with integrin β1 knockdown (a gift from JF Cui) and Huh7 (obtained from the Japanese Cancer Research Bank) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin-streptomycin (Invitrogen). The stellate cell line LX-2 cells (a gift from S. Friedman, Mount Sinai, New York) were grown in DMEM containing 2% FBS. Primary human hepatic stellate Cells (pHSCs) (ScienCell, USA) were maintained in the complete stellate cell medium. All cell cultures were conducted at 37 °C in a humidified atmosphere of 5% CO₂.