FIG. 2.

Dose- and time-dependent effects of SERCA inhibition on CHOP expression, caspase-3 activation, and cell death. Cell death was assayed in real-time by propidium iodide incorporation in MIN6 cells. A: Images illustrating the progressive propidium iodide incorporation in a field of MIN6 cells exposed to 1 μmol/l thapsigargin (Tg). B: Representative time course of cell death in response to various concentrations of thapsigargin. ○, Control; ▪, 0.01 μmol/l thapsigargin; ▴, 0.1 μmol/l thapsigargin; •, 1 μmol/l thapsigargin. C: Dose dependence of the thapsigargin-induced MIN6 cell death, quantified as the area under the curves (IAUC) of the first 24 h of the propidium iodide incorporation profiles (n = 3). D: Induction of CHOP (∼31-kDa band) and cleaved caspase-3 (∼17- to 19-kDa band) in MIN6 cells cultured for 24 h in DMEM containing 25 mmol/l glucose and increasing concentrations of thapsigargin (n = 3). E: Representative real-time imaging of caspase-3 activation in living MIN6 cells using the MiCy-DEVD-mKO FRET probe. The loss of FRET/MiCy intensity ratio, observed in the cell marked as number one, between the time points marked (a) and (b), results from cleavage of the DEVD caspase-3 target sequence. The cells were imaged for a period of 5 h, during which caspase-3 was activated in 5 of 16 (31%) thapsigargin-treated cells and in 1 of 10 (10%) control cells. (Please see http://dx.doi.org/10.2337/db07-1762 for a high-quality digital representation of this figure.)