FIG. 7.

Time-dependent effects of ER Ca²⁺ depletion on mitochondria. Mitochondrial membrane potential was monitored by flow cytometry analysis of TMRE-stained MIN6 cells. A: Representative histograms illustrating that SERCA inhibition rapidly induces mitochondrial hyperpolarization followed later (>24 h) by the collapse of mitochondrial polarization. For quantification, depolarized, intermediate, and hyperpolarized cell populations were defined as indicated in the first panel. CCCP-treated cells are shown as a control for mitochondrial depolarization. B: Quantification of the time and treatment dependence of the fraction of cells in the depolarized and hyperpolarized mitochondrial states. (n = 12 for CCCP, n = 3–4 at each time point for all other treatments; #P < 0.05 vs. control at the same time point, *P < 0.05 vs. thapsigargin alone at the same time point.) ▪, depolarized; □, hyperpolarized. C: Western blots of cleaved caspase-9 levels 24 or 48 h following treatments as indicated. Cleaved caspase-9 normalized to actin (in arbitrary units): 24 h, control 0.42 ± 0.17 vs. 1 μmol/l thapsigargin, 1.01 ± 0.17, P < 0.05, n = 4; 48 h, control 0.62 ± 0.13 vs. thapsigargin, 2.20 ± 0.48, P < 0.05, n = 4. D: Example of mitochondrial Ca²⁺ responses in a MIN6 cell following mobilization of ER Ca²⁺ by 100 μmol/l carbachol or 1 μmol/l thapsigargin.