Changes in cytosolic Ca²⁺ ([Ca²⁺]_cyt) were imaged with FURA-2. HCT116 cells were treated with 0.05% DMSO or 125 ng/ml mycolactone (MYC) for 1 h (A), 6 h (B) and 18 h (C) before FURA-2 loading and Ca²⁺ imaging. To avoid Ca²⁺ entry into the cells, experiments were carried out in the absence of external Ca²⁺ (zero [Ca²⁺]_ext). Thapsigargin (1 µM TG) was added to the bath solution to unmask Ca²⁺ leak from ER (A–C). The area under the curve (AUC) of the TG-induced Ca²⁺ transients was used as a measure of the mobilisable Ca²⁺ from intracellular TG-sensitive stores (D). The total amount of intracellular mobilisable Ca²⁺ was estimated by treating cells with ionomycin (10 µM IONO) (E). The AUC of the IONO-induced Ca²⁺ transients reflect the total amount of Ca²⁺ that is stored within the cells (F). The number of cells imaged in the experiments is given within the graph bars in D and F. Mean cell survival of HCT116 cells incubated for 18 h with 125 ng/ml mycolactone, stained with propidium iodide and Hoechst 33342 and imaged by confocal microscopy (G). Values represent the mean of three independent experiments. Data is presented as means ± SEM; n.s., non-significant; *** P < 0.001.