To evaluate the cytotoxic effect of sev, SH-SY5Y cells were stimulated with 4.1% sev for different durations (0, 2, 4, 6, or 12 h).
To confirm if sev induces neuronal cell death via ferroptosis, SH-SY5Y cells were treated with ferrostatin-1 (Fer-1; 10 µM; a ferroptosis inhibitor) from Sigma-Aldrich, (St. Louis, MO, USA) or chelating agent [20 µM; deferoxamine mesylate (DFOM)] from Sigma-Aldrich (St. Louis, MO, USA) for 12 h, followed by incubation with 4.1% sev for 6 h.
To verify whether ACSL4 contributes to sev-induced ferroptosis via AMPK/mTOR signaling, SH-SY5Y cells were cultured in DMEM/F12 medium containing compound C (10 µM) for 12 h after si-ACSL4 or si-NC transfection, and then exposed to 4.1% sev for 6 h.
To confirm if sev induces neuronal cell death via ferroptosis, SH-SY5Y cells were treated with ferrostatin-1 (Fer-1; 10 µM; a ferroptosis inhibitor) from Sigma-Aldrich, (St. Louis, MO, USA) or chelating agent [20 µM; deferoxamine mesylate (DFOM)] from Sigma-Aldrich (St. Louis, MO, USA) for 12 h, followed by incubation with 4.1% sev for 6 h.
To verify whether ACSL4 contributes to sev-induced ferroptosis via AMPK/mTOR signaling, SH-SY5Y cells were cultured in DMEM/F12 medium containing compound C (10 µM) for 12 h after si-ACSL4 or si-NC transfection, and then exposed to 4.1% sev for 6 h.