Cytosolic and Mitochondrial ATP Quantification

Cytosolic and mitochondrial ATP levels were quantified as described^{27 (link)}. Cells were transiently transfected with plasmids carrying the bioluminescence energy transfer (BRET)-based ATP biosensor BTeam without targeting signal sequence (for cyto-ATP determination) or targeted to mitochondria (for mito-ATP determination) using TurboFectin reagent (OriGene). The plasmids were a kind gift of Hiroshi Imamura, University of Kyoto. 48 h later, the cells were incubated for 30 min in phenol red-free medium supplemented with 30 μM NanoLuciferase (NLuc) inhibitor to avoid disturbance from the BTeam released from dead cells. Afterwards, NLuc substrate (Promega) was added to the medium and the plate incubated for 20 min. Subsequently, luminescent emissions from the cells were measured at 37 °C at 520/560 nm (Yellow Fluorescent Protein (YFP) emission) and at 430/470 nm (NLuc emission) using a Tecan Spark® Multimode Microplate Reader.