SH-SY5Y Neuroblastoma Cell Culture and Treatment

SH-SY5Y neuroblastoma cells (human, ECACC; Sigma Aldrich, St. Louis, MO, USA) were used in this study. Selected cell line is frequently used as a reliable model for studying neurotoxicity of drugs [48 (link),49 (link)]. SH-SY5Y cells were incubated at a density of 4 × 10⁶ cells/well in 6-well culture plates in Ham’s F-12 Nutrient Mixture (Thermo Fisher, Waltham, MA, USA) and minimum essential medium (MEM) (Sigma Aldrich, St. Louis, MO, USA) (mixed in ratio 1:1) medium supplemented with penicillin (100 U/mL), streptomycin (100 µg/mL), and L-glutamine (2 mM) without fetal bovine serum at 37 °C in saturated humidity atmosphere containing 5% CO₂. Cells were not treated (control) or treated with bortezomib 50 nM/l (Cell Signalling Technology, Danvers, MA, USA) and collected after 24 h of incubation [50 (link),51 (link)]. Cells were then subjected to mRNA, miRNA, and protein isolation.