Recombinant epidermal growth factor

Cells were purchased from the American Type Culture Collection (ATCC). MCF7 was cultured in Dulbecco's modified Eagle's medium (DMEM) (low glucose; Corning) supplemented with 10% FBS (HyClone). T47D and BT549 were grown in RPMI 1640 (Corning) with 10% FBS and 0.2 U/ml insulin. HEK293T cells were cultured in DMEM (high glucose) with 10% FBS. MDA‐MB‐231 was maintained in Leibovitz's L‐15 medium (Gibco) with 10% FBS. MCF10A was cultured in DMEM/F12 medium supplemented with 5% horse serum, 10 mg/ml insulin, 0.5 mg/ml hydrocortisone, 20 ng/ml recombinant epidermal growth factor, 100 ng/ml cholera toxin, and 1% antibiotics (Lonza). Cells were maintained in a humidified incubator with 5% CO₂ at 37°C.

Freshly isolated primary human hepatocytes were provided by Regenerative Medicine and Experimental Surgery, Hannover Medical School, Hannover, as reported.⁽¹²⁾ Mouse primary hepatocytes were isolated from mouse liver as described.⁽⁸⁾ Briefly, mice were anesthetized, and their livers were perfused with Liberase (Roche). After perfusion, livers were disintegrated mechanically before collecting hepatocytes by low‐speed centrifugation. Nonparenchymal cells were removed by discarding the supernatant. For all in vitro transfection experiments, we used Percoll density gradient‐purified mouse hepatocytes to achieve high transfection efficiency. We seeded 100,000 primary hepatocytes per well of a collagen‐coated 12‐well plate (TPP). Twelve hours after seeding, hepatocytes were transfected with 25 nM or 50 nM miR‐125b‐5p mimic, miR‐125b‐5p inhibitor, control scramble (Qiagen), or 100 nM ABTB1 small interfering RNA (siRNA) (Qiagen) using Targefect reagent in the presence of virofect enhancer (Targeting Systems). Transfected hepatocytes were cultured in hepatocyte culture medium (Lonza) containing recombinant epidermal growth factor (an inducer of hepatocyte proliferation), transferrin, ascorbic acid, insulin, hydrocortisone, bovine serum albumin, and gentamicin sulfate‐amphotericin.