Fig. 4. Effects of menadione on mitochondrial Ca²⁺ and membrane potential. (A) Ca²⁺ concentration in mitochondria following addition of 100 nM ACh and menadione. (B,a-d). Colocalisation of TMRE and Mito Tracker Green in pancreatic acinar cell cluster. (a) TMRE fluorescence; (b) Mito Tracker fluorescence; (c) overlay of Mito Tracker Green and TMRE; (d) transmitted light image of the same cell cluster; (e) changes in mitochondrial region membrane potential (green box) and nuclear region (yellow box) of the same cell measured with TMRE during application of menadione followed by 10 µM mCCCP. (C) Changes in mitochondrial membrane potential following application of menadione alone (control cell); pre-treatment with either ZVADfmk, BAPTA or bongkrekic acid (BA). (D) Changes in mitochondrial membrane potential following application of 100 nM antimycin A to control cells (upper trace) and to bongkrekic acid treated cells (lower trace) are shown. (E-G). Menadione induces a cytosolic Ca²⁺ rise and mitochondrial depolarisation simultaneously. (E,F) Confocal images of a two-cell cluster loaded with TMRE (E) and Fluo-4 AM (F). (G) Changes in free Ca²⁺ concentration, as measured by Fluo-4 (green line) and fluorescence of TMRE (red line) after application of 100 nM ACh and menadione measured from mitochondrial region of the left cell (blue box).