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Figure 4.

The effects of mycolactone (MYC) on [Ca2+]ER were analysed in wild-type HCT116 cells (A, WT) and in HCT116 cells containing point mutations in Sec61α (B, D60G; C, R66K; D, S71F; E, S82Y; F, Q127K). To image [Ca2+]ER, cells were transfected with the ER Ca2+ sensor ER-GCaMP6-150. Changes in [Ca2+]ER are given as normalised fluorescence (F/F0). Cells were treated with 0.05% DMSO or 125 ng/ml mycolactone for 6 h before Ca2+ imaging. Thapsigargin (1 µM TG) was used to induce ER Ca2+ depletion. Accordingly, the speed in the decrease in [Ca2+]ER is a measure of the Ca2+ leak from ER and was calculated as the time to 50% decrease in the normalised fluorescence F/F0 (G, t1/2). The number of cells is given within the graph bars in E. Data is presented as means ± SEM; * P < 0.05; *** P < 0.001.

Mycolactone-resistant mutations in Sec61α abolish the effects of mycolactone on the Ca²⁺ leak from ER.

The effects of mycolactone (MYC) on [Ca²⁺]_ER were analysed in wild-type HCT116 cells (A, WT) and in HCT116 cells containing point mutations in Sec61α (B, D60G; C, R66K; D, S71F; E, S82Y; F, Q127K). To image [Ca²⁺]_ER, cells were transfected with the ER Ca²⁺ sensor ER-GCaMP6-150. Changes in [Ca²⁺]_ER are given as normalised fluorescence (F/F₀). Cells were treated with 0.05% DMSO or 125 ng/ml mycolactone for 6 h before Ca²⁺ imaging. Thapsigargin (1 µM TG) was used to induce ER Ca²⁺ depletion. Accordingly, the speed in the decrease in [Ca²⁺]_ER is a measure of the Ca²⁺ leak from ER and was calculated as the time to 50% decrease in the normalised fluorescence F/F₀ (G, t_1/2). The number of cells is given within the graph bars in E. Data is presented as means ± SEM; * P < 0.05; *** P < 0.001.

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