Determination of Nrf2 activation and qPCR quantification of ARE gene expression

Primary corticostriatal neuronal co-cultures were prepared from E18 Sprague-Dawley rat or C57Bl/6 mouse embryos of either gender as previously described¹⁶. For luciferase reporter assays, the Cignal Antioxidant Response Reporter kit (Qiagen) was used. The 5xARE luciferase reporter mixture at 40:1 luciferase:Renilla plasmid was transfected into cortical and striatal neurons separately using an Amaxa electroporation device (Lonza). After electroporation, neurons were pooled and immediately plated into 96-well plates containing mature glial cultures. After culturing for 96 h, compounds were added at the indicated concentrations for 7 or 24 h prior to harvesting using Dual-Glo Luciferase Assay System protocol and reagents (Promega). Dual-wavelength luminescence was detected using a SpectraMax L microplate reader (Molecular Devices). Luciferase values were normalized to the internal Renilla control and fold-expression over the DMSO-only treatment control was calculated. At least 3 independent experiments were done using 4–6 biological replicates.

For qPCR quantification of ARE target gene expression levels, cortical and striatal neurons were plated onto 96-well plates containing mature glial cultures and cultured for 96 h. Fraction 0–4 was added to cultures at the indicated concentrations for 6 h. At the end of the treatment period, cells were lysed and total RNA was isolated using Absolutely RNA mini-prep kits (Agilent Technologies/Stratagene). cDNA was generated using oligo dT primers and Superscript II reverse transcriptase (Invitrogen). Resulting cDNA was used for quantitative PCR of gene transcripts using SYBR Green Real-Time PCR Master Mix (Life Technologies) and the following mouse primers, for: Gclc (forward-5′ TGGCCACTATCTGCCCAATT-3′ and reverse-5′- GTCTGACACGTAGCCTCGGTAA-3′), Nqo1 (forward-5′-GCCCGCATGCAGATCCT-3′ and reverse 5′-GGTCTCCTCCCAGACGGTTT3′), Srx (forward-5′-GCTTCCTCTCGGGAGTCCTT-3′ and reverse-5′-CAGCAACAGCGACTACGAAGTAA-3′), and Hmox1 (forward-5′-CCTCACTGGCAGGAAATCATC-3′ and reverse-5′-CCTCGTGGAGACGCTTTACATA-3′) (Integrated DNA Technologies). For rat corticostriatal co-culture samples, qPCR primers used were as previously described⁴⁷. Each biological sample was measured in triplicate on a ViiA 7 real-time PCR instrument (Applied Biosystems); fold expression was calculated after normalization to corresponding control GAPDH levels.