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. Author manuscript; available in PMC: 2010 Jul 1.
Published in final edited form as: Prostaglandins Leukot Essent Fatty Acids. 2009 May 29;81(1):53–59. doi: 10.1016/j.plefa.2009.04.008

Figure 3.

Figure 3

EKODE induces endogenous ARE regulated genes and affects GSH levels in IMR-32 cells. (A) Effect of EKODE on NQO1 transcript levels as analyzed by quantitative real-time PCR (qPCR). IMR-32 cells were treated for 8 h, total RNA was isolated, reverse-transcribed to cDNA and subjected to TaqMan analysis. Results are the relative expression means ± standard deviation (n = 3). GAPDH was used as internal control for normalization. EKODE induced NQO1 expression at 1 and 10 μM. For comparison, NRF2 transcript levels remained unchanged. (B) Effect of EKODE on NQO1 protein levels as analyzed by immunoblot analysis. IMR-32 cells were treated with EKODE for 24 h, proteins were extracted, resolved by SDS-PAGE and subjected to immunoblot analysis for NQO1 (β-actin = control). EKODE greatly increased NQO1 protein levels at 10 μM. (C) Time-dependent changes of GSH levels in IMR-32 cells upon exposure with EKODE vs. vehicle (0.5% DMSO). Cells were treated with EKODE for the indicated times, harvested and resuspended in 5% SSA solution. Upon two freeze thaw (liquid N2/37 °C) cycles and centrifugation, the supernatant served as a source of cellular glutathione. Total glutathione (GSH, GSSG) was assessed using the Glutathione Assay Kit (Sigma). Results are the means ± standard deviation (n = 2).