2.1 Cell culture

Stable knockdowns of ELOVL5 and IGFBP6 genes were performed using RNA interference (Schwankhaus et al., 2014; Nikulin et al., 2018; 2021; Maltseva et al., 2020). DNA oligonucleotides selected for the target sequences in the ELOVL5 and IGFBP6 genes were ligated into the pLVX shRNA1 lentiviral vector (Clontech Laboratories) according to the manufacturer’s protocol. To obtain the control MDA-MB-231 cells, we used the same lentiviral vector pLVX shRNA1 containing shRNA to the Photinus pyralis firefly luciferase gene. Viral particles were obtained in the form of cell-free supernatants using transient transfection of HEK-293T cell line according to the previously described method (Weber et al., 2010; 2012). Supernatants were collected 24 h after transfection, filtered using .45 µm syringe filters and stored at −80°C. Then, 5∙10⁴ MDA-MB-231 cells were cultured in the wells of a 24-well culture plate in 0.5 mL of cell culture medium. After 24 h, 10 μL of the supernatant containing viral particles was added to the wells, and the plate was placed in a cell culture incubator for 24 h. Then the cell culture medium was changed and the cells were incubated for another 24 h. After that, the selection with 1 μg/mL puromycin (Gibco) was carried out for 2 weeks.

Cells were cultured in a complete culture medium consisting of DMEM 4.5 g/L glucose (Gibco) supplemented with 10% vol. fetal bovine serum (Gibco) and 1% vol. antibiotic-antimycotic solution (Gibco). The cells were incubated in a cell culture incubator at 37°C, 5% CO₂ (Sanyo). Subcultivation was performed every 2–3 days using trypsin-EDTA solution (PanEco). Cells were counted after trypan blue (Gibco) staining using Countess automated cell counter (Invitrogen) according to the manufacturer’s protocol.