Journal: Cell Death & Disease

Article Title: The mycotoxin phomoxanthone A disturbs the form and function of the inner mitochondrial membrane

doi: 10.1038/s41419-018-0312-8

Figure Lengend Snippet: a Live measurement of the effect of PXA on uncoupled cellular respiration in intact Ramos cells. Cells were first treated with 1 µM CCCP to uncouple respiration and then with either 10 µM PXA or 0.1% v/v DMSO (vehicle control) to test for inhibition. Measurement of extracellular [O 2 ] was performed using an oxygraph. b Comparative measurements of the effect of PXA and other compounds on basal and uncoupled respiration in Ramos cells. Respiration was uncoupled by CCCP (1 µM). PXA (10 µM) was compared to the complex-specific ETC inhibitors rotenone (Rot; complex I; 10 µM), thenoyltrifluoroacetone (TTFA; complex II; 10 µM), antimycin A (AmA; complex III; 10 µM), azide (NaN 3 ; complex IV; 1 mM) and oligomycin A (OmA; complex V; 10 µM). Measurement was performed in a microplate reader using the fluorescence-based MITO-ID® Extracellular O2 Sensor Kit (High Sensitivity) (Enzo). c Comparative measurements of the effect of PXA and other compounds on ATP levels in Ramos cells after 120 min of treatment. PXA (10 µM) was compared to complex-specific ETC inhibitors (see above) as well as CCCP (1 µM). Measurement was performed in a microplate reader using the luminescence-based Mitochondrial ToxGlo™ Assay (Promega). Cells were incubated in full growth medium containing either glucose or galactose as the only available sugar. Galactose alone forces the cells to resort exclusively to OXPHOS for ATP synthesis. d Live measurement of mitochondrial respiration in Ramos cells permeabilized by digitonin (Digi; 5 µg/ml). The effect of PXA (first 1 µM, then increased to 10 µM as indicated) was compared to that of the known complex I inhibitor rotenone (Rot; 1 µM). To specifically induce complex II and III of the electron transport chain, succinate (Succ; 10 mM) and duroquinol (Duro; 1 mM) were used, respectively. Measurement of extracellular [O 2 ] was performed using an oxygraph

Article Snippet: This measurement was performed using the MITO-ID® Extracellular O2 Sensor Kit (High Sensitivity) (Enzo Life Sciences, Lörrach, Germany; #51045) according to manufacturer’s instructions.

Techniques: Inhibition, Fluorescence, Incubation