Dmem 4 500 mg l glucose

The experiments were performed using two TNBC cell lines Hs578T and MDA-MB-231, cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) 4,500 mg/L glucose (Sigma-Aldrich, St Louis, MO, USA) and RPMI-1640 (Sigma-Aldrich) medium, respectively. They were supplemented with 10% fetal bovine serum (Sigma-Aldrich), 1% l-glu-tamine solution, and 1% penicillin–streptomycin (both from Sigma-Aldrich). For gene silencing, we used siRNA-p53 and siRNA-TNF from Ambion (Austin, TX, USA). siPORT™ NeoFX™ Transfection Agent (Life Technologies, Carlsbad, CA, USA) was used to perform the transfection, according to the manufacturer’s protocol. The transfection was done simultaneously with cell plating. For further RT-PCR studies, we used a density of 5×10⁵ cells/well in a total volume of 2 mL Opti-MEM I (Thermo Fisher Scientific, Waltham, MA, USA). For each well, we used 5 μL transfection agent and a final concentration of 40 nM siRNA. The cells were treated simultaneously with both siRNAs for p53 and TNF gene silencing. As a control, we used a scramble siRNA. Each experiment was performed in triplicate. Cells were incubated for 24 hours in a 5% CO₂ incubator at 37°C and then lysed to isolate total RNA.