Fig. 5. IP3R-mediated ER Ca²⁺ release induces transient SER tubule-mitochondria dissociation. MDCK cells were treated with 10 µM ATP for up to 5 minutes. Treated MDCK cells were fixed at time 0 (A-C'), 2 (D-F') or 5 (G-I') minutes and then double immunofluorescently labelled for 3F3A and mtHSP70. Merged images (C,C',F,F',I,I') show 3F3A (red) and mitochondria (green), co-distribution in yellow. C', F' and I' show details of the boxed regions from images C, F and I, respectively. (J) At various times of ATP treatment without (solid lines) or with xestospongin C (XeC) pre-treatment (dashed lines), the extent of dissociation of 3F3A-labelled SER tubules from mitochondria was quantified (blue, right Y-axis) and the corresponding average [Ca²⁺]_cyt determined (red, left Y-axis). The data represent the average (±s.e.m.) of three independent experiments and [Ca²⁺]_cyt values the average of five Fura-2-AM experiments. Bars, 20 µm (H); 2 µm (I).