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.