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. 2017 Sep 19;24:147–158. doi: 10.1016/j.ebiom.2017.09.022

Fig. 1.

Fig. 1

Live-cell imaging of mitochondrial ATP under hypoxia and effects of small-molecule compounds. (a) Representative images of fluorescence resonance energy transfer (FRET) signal in HL-1 cardiomyocytes stably expressing either cyto-ATeam (upper image) or mito-ATeam (lower image). Scale bars, 20 μm. (b) Quantified FRET ratio (YFP/CFP) in response to 10 μM oligomycin A (OA) and 10 mM 2-deoxyglucose (2-DG) treatment in HL-1 cardiomyocytes stably expressing either cyto-ATeam (upper panel) or mito-ATeam (lower panel). Data are presented as means ± SD (n = 3 biologically independent samples). The arrow heads indicate the time point when vehicle or OA + 2-DG was added. (c) Representative images of FRET signal in HL-1 cardiomyocytes stably expressing either cyto-ATeam (upper panel) or mito-ATeam (lower panel) under hypoxia (1% O2) followed by reoxygenation (21% O2). Scale bars, 20 μm. (d) Quantified FRET ratio in HL-1 cardiomyocytes stably expressing either cyto-ATeam (black line) or mito-ATeam (red line) under 2-h hypoxia (1% O2) followed by 2-h reoxygenation (21% O2). Data are presented as means ± SD (n = 3 biologically independent samples). (e) HL-1 cardiomyocytes stably expressing mito-ATeam were treated with compounds typically for 24 h, and subjected to FRET imaging under hypoxia followed by reoxygenation as shown in (d). (f) Effects of cyclosporin A, mito-TEMPO, deferoxamine, Compound 968, N-acetyl cysteine (NAC), dexrazoxane, diazoxide, cariporide, or verapamil on mitochondrial ATP under hypoxia are shown. Data are presented as means ± SD (n = 3 biologically independent samples).