Anti human postn antibody

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.

When LX-2 or pHSCs cells were grown to 80% confluence in 25-cm² flasks, cells were washed with PBS, the medium was exchanged with fresh culture medium and incubated for 24 h. The CM of HSCs was collected and preserved in −80 °C, followed by centrifugation with 3000 rpm for 15 min to remove debris. Culture medium without cells incubated for 24 h in culture flasks served as the control.
For neutralizing POSTN in the CM of HSCs, CM was pre-incubated with anti-human POSTN antibody (2,500 ng/ml) (Abcam) or its control antibody for 1 h at room temperature before it was subjected to subsequent experimental use.
For individual experiments, HSCs were exposed to 10 mM metformin (Sigma-Aldrich) or DMSO for 12 h, then the medium was discarded and replaced with fresh medium. After 24 h, CM from metformin-treated HSCs was collected for subsequent experiments.