Figure 7

Expression of insulin 2 (C96Y)-EGFP induces apoptosis. A. Clone #4S2 cells were treated or not with 2 μg/ml doxycycline for 72 h, fixed and imaged by differential interference contrast microscopy. Potentially apoptotic cells with a rounded morphology are indicated (arrows). B-D. Clone #4S2 cells were treated with 2 μg/ml doxycycline for the indicated times. B. Following the treatments, the cells were lysed and apoptosis was measured using a cell death detection ELISA kit (Roche) as described in the methods. Shown are the mean ± SE of 4 independent experiments. *p < 0.05. C. Cells were washed in PBS, lysed and equal amounts of protein were resolved by NuPAGE and immunoblotted using antibodies to cleaved caspase-3 and γ-tubulin. D. Cells were fixed and labeled with Alexa Fluor 647 dye-labeled anti-BrdU antibody. Cells were analyzed by flow cytometry; the FL1 laser excites GFP, whereas FL4 excites Fluor 647. Cells in the upper left and right quadrants were classified as mutant insulin expressing, cells in the upper and lower right quadrants were classified as apoptotic. The total number of cells examined is shown below each chart, and the percentage of cells in each quadrant is indicated.