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Fig. 6. Effect of CAI treatment on [Ca²⁺]_m dynamics in digitonin-permeabilized HEK-293 cells. (A) Mean Rhod-2/MitoTracker Green ratio fluorescence changes for suspensions of permeabilized HEK cells pre-treated with 30 µM Ca²⁺. Cells were treated at time 0 with vehicle (closed circles) or 10 µM CAI (open circles) and fluorescence changes were monitored by multi-well fluorescence plate reader. (B) Changes in [Ca²⁺]_m in permeabilized HEK cells in response to intracellular saline solutions containing specific set levels of [Ca²⁺] (3-3000 µM) measured as in Fig. 3. (C) Relationship between the concentration of Ca²⁺ added and the mean change in mitochondrial fluorescence in permeabilized cells. Mitochondrial fluorescence was measured 300 seconds after adding Ca²⁺. , 10 µM CAI-treated cells; , vehicle-treated cells. (D) Effect of pre-treatment with CAI on change in [Ca²⁺]_m induced by adding 30 µM Ca²⁺. Representative traces are shown, demonstrating an attenuation and/or loss of mitochondrial fluorescence compared with the control. (E) Representative digital fluorescence images (a-e) of HEK cells loaded with 2 µM mixture of Rhod-2, Rhod-FF and Rhod-5N dyes (1:1:1). Cells were permeabilized with 10 µM digitonin for 3 minutes in nominal Ca²⁺ and then challenged (arrow) with the saline solution containing 300 µM free Ca²⁺. The trace is the mean time-dependent change in fluorescence measured from individual puncta (see insets). Ca²⁺-containing solutions were applied locally (about 50-100 µm from the field of view) using a pressure-driven perfusion system. Bars, 10 µm.

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