Neuroblastoma Cell Line Differentiation

The SH-SY5Y human neuroblastoma cell line was obtained from Sigma-Aldrich (cat. n° 94030304) (St. Louis, MO, USA). Cells were expanded in a growth culture medium composed of high glucose Dulbecco’s Modified Eagle’s medium (DMEM), supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, 50 U/mL of penicillin, and 50 μg/mL of streptomycin, and cultured at 37 °C with 5% CO₂ as previously reported [31 (link)]. Cell differentiation was induced by reducing serum levels of the medium to 1% with 10 µM retinoic acid (RA) for seven days prior to treatments [32 (link)]. SF (LKT Laboratories, Minneapolis, MN, USA), EGCG and PB (Sigma-Aldrich, St. Louis, MO, USA) were dissolved in DMSO, and 10 mM stocks were kept at −20 °C until use. Differentiated SH-SY5Y cells were treated with 1 μM SF, 2.5 μM EGCG, and 0.5 μM PB and SEP (1 μM SF + 2.5 μM EGCG + 0.5 μM PB,) for 24 h or 6 h according to the different experiments. Oxidative stress was induced, as previously reported [33 (link)], by exposing cells to 700 µM H₂O₂ in 1% FBS DMEM.