Evaluating Nrf2-Mediated Transcriptional Response

Wild-type (WT) and Nrf2-disrupted mouse embryonic fibroblasts (MEF) that were established according to previously published protocols [23 (link)] were seeded at 5 × 10⁵ cells/well in a 6-well plate. Cells were cultured in Iscove’s Modified Dulbecco Medium with 10% FBS. Cells were treated with TTCA (0.5 – 4 μM) or 10 μM SFN and were harvested 20 hours later. RNA was isolated using TRIzol (Invitrogen, Carlsbad, CA) and quantified using UV spectrophotometry at 260 nm. An absorbance ratio of 260/280 was utilized to determine the purity of RNA. RNA integrity was confirmed by gel electrophoresis and cDNA was synthesized using 1 μg of RNA with the qScript system (Quanta Biosciences, Beverly, MA). Real time PCR was performed on a Bio-Rad My-IQ with SYBR Green (Bio-Rad, Hercules, CA). PCR efficiency was determined using a standard curve method and the Pfaffl method was used for quantification of fold changes [24 (link)]. Primer sequences for Nqo1 and Gapdh were as previously published [25 (link)]. SFN was obtained from LKT laboratories (St Paul, MN).