Primary mouse HSCs were isolated using sequential pronase-collagenase digestion. Briefly, livers were perfused in situ with Hanks’ balanced salt solution (HBSS) buffer without Ca2+ and Mg2+ (Thermo Fisher Scientific) supplemented with 0.5 mM EGTA for 5 min. Then, liver tissue was perfused with pronase E (0.7 mg/ml; Roche) for 5 min and collagenase P (0.25 mg/ml; Roche) for 6 to 8 min, respectively, at a flow rate of 5 ml/min in HBSS buffer containing Ca2+. After excision of the liver, the liver was digested in vitro for 15 min in HBSS containing 1% deoxyribonuclease I (DNase I; Roche). HSCs were purified from the remainder of nonparenchymal cells and hepatocyte-derived debris by floatation through 9% (w/v) OptiPrep (Axis-Shield PoC AS, Oslo, Norway) in HBSS buffer. The isolated HSCs were cultured in Dulbecco’s modified Eagle’s medium (DMEM)/F-12 (Thermo Fisher Scientific) supplemented with 10% FCS. For TNF-α treatment, primary HSCs were isolated from Rhbdf2+/− and Rhbdf2−/−, the isolated HSCs were cultured in DMEM/F-12 supplemented with 10% FCS for 3 days, and then, the cells were starved in serum-free DMEM/F-12 overnight. Next day, the cells were treated with and without TNF-α (50 ng/ml) in DMEM/F-12 supplemented with 10% FCS for 24 hours.

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