D2141

HEK 293T cells, HeLa cells and mouse BNL CL2 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with high glucose (11965, Gibco) supplemented with 10% fetal bovine serum (04-001, Biological Industries) and 1% penicillin-streptomycin (15140, Gibco). Cells were kept in a 37°C incubator with 5% CO₂. DON (D2141, Sigma-Aldrich) and cytidine were dissolved in water. Mycophenolic acid (M3536, Sigma-Aldrich), guanosine, 3′-deazauridine (sc-394445, Santa Cruz Biotechnology), LY294002 (9901S, Cell Signaling) and rapamycin (R0395, Sigma-Aldrich) were dissolved in DMSO (D2650, Sigma-Aldrich). Chemicals were added in culture medium as described.

PLC/PRF/5, SK-Hep1 were obtained from the American Type Culture Collection (Manassas, VA), MHCC-97H was from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). All cell lines were confirmed free of mycoplasma (MycoAlert PLUS kit; Lonza, Basel, Switzerland) and cell authentication was performed by short tandem repeat profiling (Beijing Microread Gene Technology Co., Beijing, China). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Grand Island, NY, USA) supplemented with 10% FBS (Corning, NY, USA), 100 mg/mL streptomycin, and 100 IU penicillin at 37 °C containing 5% CO₂.
Cells were cultured in medium supplemented with 6-diazo-5-oxo-l-norleucine (DON, 20 μM; D2141; Sigma-Aldrich, St Louis, MO, USA), ST045849 (ST, 50 μM; MFCD03308174; Tim Tec, Newark, USA), Thiamet G (TG, 25 μM; S7213; Selleckchem, Houston, USA) or Cycloheximide (CHX, 100 μM; HY-12320; MedChemExpress, New Jersey, USA) and then collected for analysis.