*2.4. In Vitro Cell Culturing, Drug Treatment, Nuclear Factor-2 Erythroid-2 (Nrf2) Gene Silencing by Small Interfering RNA (siRNA) and Western Blot Analysis*

The mouse hippocampal HT22 and murine BV2 microglial cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with FBS (Fetal bovine serum) (10%) and penicillin/streptomycin (1%) in a 5% CO2 incubator at 37 ◦C. After attaining a confluency of 70%, the cells were pretreated for 1 h with ethanol (100 μM), followed by curcumin (2 μM) or Nrf2 siRNA for 24 h, or TAK242 (TLR4 specific inhibitor). The Nrf2 gene was knocked down with Nrf2 siRNA at a concentration of 10 μM per transfection for 36 h, as directed (SC: 37049, Santa Cruz Biotechnology, Inc., Dallas, TX, USA). The transfection was conducted with lipofectamine™2000 reagent (Invitrogen, Waltham, MA, USA) when the cells culture reached to 75–80%. The control group cells were treated with 0.01% Dimethyl sulfoxide (DMSO).
