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Hypoglycemia induces progressive down-regulation of Nrf2 expression (protein) and function in hCMEC/D3 cells.

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posted on 2015-03-25, 05:01 authored by Ravi K. Sajja, Kayla N. Green, Luca Cucullo

(A) IF and western blot analyses of BBB endothelial Nrf2 and its downstream target, NQO1, expression and distribution following 3-24h exposure to normal or hypoglycemic media (see Methods; n = 3-4/condition). Respective bands with β-actin as loading control were shown above the graphs for each time point. (B) Effects of hypoglycemia on protein expression/distribution of intracellular regulators of Nrf2, such as Siah2 (3-12h) and Keap1 (12h; B2), as assessed by IF and western blots (n = 3-4/condition). Further, a magnified view of the region represented by yellow box was provided in the inset to demonstrate the cellular localization changes of Siah2 following 12 h exposure to control or hypoglycemic conditions. (C) Real-time qRT-PCR based analysis of mRNA expression of target genes in hCMEC/D3 cells exposed to normal or hypoglycemic media (12h) (n = 4/condition). Data were expressed as mean ± SEM (% normalglycemic control for western blots) or fold change over control (mRNA expression). Images were captured at 40X (scale: 100μm) and merged with DAPI. * P < 0.05 vs. control. Experiments were repeated twice.