2 5 dimethyl celecoxib

Freshly isolated LSECs were unstable in our preliminary experiments due to spontaneous capillarisation and short survival. SK‐Hep1, an immortalised human LSECs line, can maintain endothelial phenotype persistently without reagent induction.²⁵, ²⁶ It was obtained from the Procell Life Science and Technology Co., Ltd. The cells were cultured in Dulbecco's modified Eagle's medium (DMEM, HyClone) supplemented with 10% fetal bovine serum (FBS, Biological Industries), and 100 U/ml penicillin and 100 μg/ml streptomycin (HyClone) in a humidified atmosphere at 37°C with 5% CO₂ in the air.
SK‐Hep1 cells were serum‐starved with 1% FBS for 16 hours before treatments. All treatments were performed in serum‐free DMEM medium. Celecoxib (1, 5, 10 μM, Selleck Chemical), 2,5‐Dimethyl‐Celecoxib (DMC, a structural analogue of Celecoxib with no COX‐2‐inhibiting activity, 1, 5, 10 μM, Sigma‐Aldrich), PGE2 (0.2, 1, 5 μM, MedChem Express), E‐prostanoid receptor 2 (EP2) antagonist (TG4‐155, 10 μM, MedChem Express), and ERK1/2 inhibitor (AZD6244, 1 μM, Selleck Chemical) were dissolved in dimethyl sulfoxide (DMSO, Sigma‐Aldrich). The final concentration of DMSO was 0.1% in the treated cells.

Human hepatoblastoma cell line HepG2 (HB-8065, ATCC, Manassas, VA), HepG2.2.15 (donated by the laboratory of the First Affiliated Hospital of Fujian Medical University), hepatoma cell lines Huh7 (JCRB0403, Japan), HL7702 and BEL-7404 (Shanghai Institute of Cell Biology, Chinese Academy of Sciences), MHCC97H (Fudan University Hepatology Research Institute) were maintained in Dulbecco's modified Eagle medium (DMEM, Gibco, Carlsbad, CA) supplemented with 12% foetal bovine serum (FBS, Gibco, Carlsbad, CA).
The cells were seeded at a density of 5 × 10⁵ cells/well, and DNA transfection was performed in a six-well plate using Lipofectamine 2000 (Life Technologies, Carlsbad, CA) according to the manufacturer's instructions. The transfection ratio of the Donor vector PX459 (donated by the Key Laboratory of Ministry of Education for Gastrointestinal Cancer of Fujian Medical University): sgRNA vector was 3:1, and the co-transfected cells were screened for 2 week using 1 mg/mL G418. The red fluorescent PE intensity (the signal intensity of PE channel) of the reporter cells was detected using the BD Accuri C6 PlusTM platform. The materials used in the transfection experiments were interleukin-1β (IL-1β, ab9617, Abcam, Cambridge, UK) and small molecule inhibitors MK-886 and MF63 (Cayman Chemical, Ann Arbor, MI), 1,3-benzoxazol-5-amine and 2,5-dimethyl-celecoxib (Sigma-Aldrich, St. Louis, MO).