Fig. 1: PXA causes an increase of [Ca²⁺]_cyt and a release of [Ca²⁺]_mito but not [Ca²⁺]_ER.

a Live measurement of the effect of PXA (10 µM) on [Ca²⁺]_cyt in Ramos cells, where DMSO (0.1% v/v) was used as vehicle control and ionomycin (IM; 2 µM) was used as positive control, and b live measurement of [Ca²⁺]_cyt after PXA followed by thapsigargin (TG; 10 µM). Measurements were performed by flow cytometry using the Ca²⁺-sensitive fluorescent probe Fluo-4-AM (Ex 488 nm, Em 530 ± 30 nm) in the absence of extracellular Ca²⁺ by maintaining the cells in Krebs-Ringer buffer containing 0.5 mM EGTA during measurement. c, d Comparison of the effect of PXA (10 µM) and thapsigargin (TG; 1 µM) on either [Ca²⁺]_ER or [Ca²⁺]_mito as measured by the Ca²⁺-sensitive fluorescent protein CEPIA targeted to the respective organelle in HeLa cells. All traces were normalised (F/F₀) where F₀ is the starting fluorescence of each trace. e Comparison of the effect of PXA (10 µM), ionomycin (IM; 2 µM), and thapsigargin (TG; 1 µM) on [Ca²⁺]_mito in Ramos cells stably transfected with the Ca²⁺-sensitive ratiometric fluorescent protein mito-Pericam. DMSO (0.1% v/v) was used as vehicle control. F/F₀ is the ratio of fluorescence with excitation at 488 nm (high [Ca²⁺]) to 405 nm (low Ca²⁺]). f, g Live imaging and quantification of the effect of PXA (10 µM) on mPTP opening in HeLa cells as measured by mitochondrial calcein fluorescence using the calcein/cobalt quenching method. DMSO (0.1% v/v) was used as vehicle control and ionomycin (IM; 2 µM) was used as positive control. Mitochondrial calcein fluorescence was quantified. h Additional live measurement of the effect of PXA on mPTP opening in Ramos cells by the calcein/cobalt quenching method using flow cytometry