FIGURE 1.

Imaging the effects of glucose and insulin on the subcellular localization of endogenous FoxO1 or overexpressed FoxO1-EGFP in primary human and mouse β-cells. A, partially dissociated human islets seeded on coverslips for 1 day in 11 mm glucose were subsequently incubated for 16 h in 3 or 16.7 mm glucose, were formaldehyde-fixed, permeabilized, and immunostained with anti-FoxO1 and anti-insulin antibodies, and subsequently with Alexa Fluor-488- and -564-coupled secondary antibodies, while DAPI was used for nuclear staining. Cell clusters containing insulin-positive cells were imaged by confocal microscopy taking Z cross-sections at 0.2 μm spacing along the entire depth of the clusters. The three-dimensional “opacity” (Volocity^TM software) view is presented. Scale bar, 10 μm. B, ratio of fluorescence quantified within regions of interest selected in the cytosol and nucleus in the central cross-sections of single islet β cells and calculated from 25–30 individual cells. The bar shows mean ± S.E. analyzed by two-tailed t test. C, dissociated mouse islet cells plated on coverslips were transduced with FoxO1-EGFP-expressing adenovirus (20–25 MOI) and incubated for 36 h to allow expression. Following overnight incubation in 3 mm glucose, cells were exposed to either 3 or 16.7 mm glucose for the indicated time before fixation and immunostaining with anti-insulin antibody as described in A. Images show typical distribution of FoxO1-EGFP from 20–30 cells studied per condition from three independent experiments. Scale bar, 10 μm. D, ratio of fluorescence quantified from C, as described in B. Bar shows mean ± S.E. analyzed by two-tailed t test all compared to 0 time point.