Increased susceptibility of 11β-HSD1 expressing cells to H₂O₂-induced oxidative stress.

<p>H4IIE cells transiently transfected with pCDNA3 (<i>A</i>), pCDNA3 or 11β-HSD1 (<i>B, C</i> and <i>D</i>) were treated for 24 h with vehicle, 100 nM cortisol (<i>A</i>), 100 nM cortisone (<i>C</i>) or simultaneously with cortisone and 1 µM T0504 (<i>B, D</i>). The medium was replaced by assay buffer (HBSS) containing 1 g/L glucose. Single cell real-time measurements were performed on a Leica SP5 confocal microscope. After 5 min baseline adaption, cells were exposed to a final concentration of 10 µM (<i>A, B</i>) or 100 µM (<i>C, D</i>) H<b>₂</b>O<b>₂</b> and responses were compared between differentially transfected or vehicle treated cells over a period of 30 min (<i>A, B</i>) or 45 min (<i>C</i>). Data represent mean ± SEM of seven different cells for each transfection. *, <i>p</i><0.05, **, <i>p</i><0.01, ***, p<0.001, <b><i>p</i></b>-value was obtained using one-way ANOVA followed by Bonferroni post-tests compared with pcDNA3 transfected cells. To analyze the total cell population (<i>D</i>), H4IIE cells 4,000,000 cells/mL were resuspended in assay buffer. Suspensions of cells (100 µL) treated either with cortisone or cortisone and T0504 were transferred into a 96-well plate, centrifuged for 2 min at 180× g and challenged by adding 100 µL assay buffer containing 100 µM H<b>₂</b>O<b>₂</b>. Fluorescence was immediately measured after adding H₂O₂ and data were collected every 27 s at 37°C for 9 h. One of three representative experiments is shown (<i>D</i>).