Crif1 Regulates Neuronal Oxidative Stress

SH-SY5Y, a human neuroblastoma cell line, and HT22, an immortalized mouse hippocampal neuronal cell line, were cultured as previously described.^{55 (link)} Cells were transfected with cDNA for Crif1 (gifted from Dr. Shong (Chungnam University, Korea)), Sp1 (hMU003598; provided from Korea Human Gene Bank, Medical Genomics Research Center, KRIBB, Korea), Sp1 K16A or Mito-DsRed using Lipofectamine and Plus reagents (Invitrogen, Carlsbad, CA, USA) and Crif1 siRNA (Santa Cruz Biotechnology, Santa Cruz, CA, USA) using RNAiMax reagents (Invitrogen) for 48 h according to the manufacturer's instructions. Cells were grown to 60% confluency and treated with vehicle (dimethyl sulfoxide) or Aβ_1-42 (American Peptide, Sunnyvale, CA, USA), Aβ_42–1 (Bachem, Bubendorf, Switzerland) and various reagents; MG132 (10 μM; M7449, Sigma-Aldrich, St. Louis, MO, USA), 3-MA (2 mM; M9281, Sigma-Aldrich), bafilomycin (5 nM; B1793, Sigma-Aldrich), NAC (1 mM; A7250, Sigma-Aldrich), apocynin (10 μM; A10809, Sigma-Aldrich), DPI (10 μM; D2926, Sigma-Aldrich), and H₂O₂ (216763, Sigma-Aldrich).