4.2. Lentivirus Production

HEK293T cells were seeded in a 10-cm dish at a density of 5 × 10⁶ cells/dish and incubated overnight. The cells were transfected with 5 μg of the short hairpin RNA (shRNA) lentiviral vector plasmids shRNA2 and shRNA4 (Merck KGaA, Darmstadt, Germany, both targeting AHCY, respectively, and helper plasmids psPAX2 (3.75 μg) and pMD2.G (1.25 μg) using Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s instructions. For the production of control cells, we used SHC016 non-target shRNA plasmid (Merck KGaA, Darmstadt, Germany), in combination with psPAX2 and pMD2.G plasmids. After 24 h, the transfection medium was replaced with fresh medium. The supernatant containing functional lentiviral particles was collected 48 h and 72 h post-transfection, pooled, and filtered through a 0.45 μm syringe filter (Merck KGaA, Darmstadt, Germany).