2.6 Cell culture, isolation and treatment
LX-2 cells were received as a generous gift from Scott Friedman (Mount Sinai School of Medicine, New York). Cell lines were routinely tested for mycoplasma. LX-2 cells supplemented with 10% FBS and 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA). Upon reaching 60–80% confluences, the cells were starved in serum‐free DMEM for 24 hr prior to treatment with 2 ng·ml−1 TGFβ1 and/or PB (dissolved in DMSO). Primary murine HSCs were isolated from the livers of male C57BL/6J mice mice aged 6-10 weeks according to a reported protocol that includes the following steps: in situ pronase/collagenase perfusion of mouse liver, perfused livers were minced, filtered through 70 µM cell strainer (BD Bioscience), and centrifuged at 50 g for 3 min to separate hepatocytes. HSCs were isolated according to the previously published method(Mederacke, Dapito, Affo, Uchinami & Schwabe, 2015), the supernatant was further centrifuged at 500 g for 10 min, resuspended in density gradient-based Nycodenz, and centrifuged at 1400 g for 17 min. HSCs were collected from the interface. Cells were cultured in DMEM (high glucose) containing 10% FBS and 1% antibiotics. Mouse pHSCs were plated in untreated flasks and can exhibit a fibroblast‐like morphology after 7 days. Therefore, they did not need FBS‐free starvation or TGFβ1 stimulation. HEK293T cells (RRID: CVCL_0063) were cultivated in DMEM with 10% FBS and 1% antibiotics. All these cells were incubated at 37°C in a 5% CO2 atmosphere. LX‐2 and HEK293T cells were transiently transfected with plasmids or siRNAs using the Lipofectamine 3000 reagent (Invitrogen, 11668019) and Opti‐MEM serum‐free medium (Invitrogen, 1930104) according to the manufacturer’s instructions.