Cignal antioxidant response reporter luc kit

Cell culture reagents including RPMI 1640 medium supplemented with glutamine but without phenol red, heat-inactivated fetal bovine serum (FBS), penicillin/streptomycin (10,000 units/mL and 10,000 μg/mL, respectively), 0.25% typsin-EDTA, 10 mM non-essential amino acids in Minimal Essential Medium, Opti-MEM I Reduced Serum Medium supplemented with L-glutamine but without phenol red, Hank’s Balanced Salt Solution (HBSS) with calcium and magnesium but without phenol red, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and 6-carboxy-2’,7’-dichlorodihydrofluorescein diacetate (carboxy-H2DCFDA) were from Gibco/ThermoFisher Scientific (Carlsbad, CA). Tert-butyl hydroperoxide (TBHP; 70% in water), deferoxamine mesylate salt (DFO), N,N’-diphenyl-p-phenylenediamine (DPPD), butylated hydroxyanisole (BHA), indomethacin, dimethyl sulfoxide (DMSO), Tween 20, and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO). The inhibitor PD169316 was purchased from EMD Millipore (Billerica, MA). The inhibitors U0126 and SP600125 were from Enzo Life Sciences (Farmingdale, NY). QIAshredder columns, RNeasy kits, RT² First Strand Kits, RT2 SYBR Green qPCR Mastermix, prostaglandin-endoperoxide synthase 2 (PTGS2) primers, and Cignal Antioxidant Response Reporter (luc) Kit were purchased from Qiagen (Germantown, MD).

The transcriptional activity of NRF2 was assayed using the Cignal Antioxidant Response Reporter (luc) Kit (#CCS-5020L, Qiagen) according the manufacturer’s instructions.

Unless otherwise specified, Cignal Antioxidant Response Reporter (luc) Kit (CCS-5020L) was from Qiagen and used according to the manufacturer’s protocol. In specific cases as indicated in the figure legends, a mixture of luciferase plasmids (pGL4.37, E364A from Promega and pGL4.75, E693A from Promega, used as a 20:1 pGL4.37/pGL4.75 mixture) was used instead of the plasmid mixture in the commercially available Qiagen kit. Following 18 h of treatment, cells were lysed with lysis buffer (Promega). Transcription activity was determined by the expression of firefly luciferase and was normalized to the Renilla luciferase levels by using a dual luciferase reporter assay kit (Promega). Data collection was done on a BioTek cytation3 multi-mode microplate reader.