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

<p><b>(A)</b> 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122358#sec002" target="_blank">Methods</a>; n = 3-4/condition). Respective bands with β-actin as loading control were shown above the graphs for each time point. <b>(B)</b> Effects of hypoglycemia on protein expression/distribution of intracellular regulators of Nrf2, such as Siah2 (3-12h) and Keap1 (12h; <b>B2</b>), 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. <b>(C)</b> 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. * <i>P</i> < 0.05 vs. control. Experiments were repeated twice.