Dmem f12

Huh7, Huh7/sorafenib‐resistant (SR), and PLC5 cells were kindly provided by D‐L Ou, Graduate Institute of Oncology, College of Medicine, National Taiwan University, Taipei, Taiwan. PLC5/SR cells were established by long‐term exposure to sorafenib (LC Laboratories, Woburn, MA, USA) at a low dose (5 μm), which was increased to a higher dose (20 μm) over 3 months. HEK‐293T cells were obtained from the American Type Culture Collection. The HEK‐293T, Huh7, PLC5, Huh7/SR, and PLC5/SR cells were grown in Dulbecco's modified Eagle's medium (DMEM)/F12 (HiMedia, MUM, IND). sorafenib (5 μm) was added to maintain sorafenib resistance in the Huh7/SR and PLC5/SR cells. These cells were supplemented with 10% fetal bovine serum (Gibco, Waltham, MA, USA) and 1% penicillin/streptomycin (GeneDireX, Las Vegas, NV, USA) at 37 °C under 5% CO₂.

Mouse Leydig (TM-3) cells obtained from the American Type Culture Collection (ATCC®, USA) were cultured using a standard sterile cell culture technique. Medium consisted of Dulbecco’s Modified Eagle’s Medium F12 (DMEM/F12, 1:1 Mixture, Himedia) with 10% Fetal Bovine Serum (FBS; Gibco TM) and 1% Penicillin-Streptomycin (Sigma). The cells were maintained at 37°C in humidified air containing 5% CO₂. Routinely, TM-3 cells were cultured in T-75 culture flasks containing 15–18 ml complete cell culture medium. They were sub-cultured, when 80–90% confluent, cells were harvested by using 0.25% Trypsin-EDTA 1X Solution (Himedia) and were used to conduct experiments.