Figure 1.
ETC inhibition decreases the ATP:ADP ratio and arrests mitochondria. (A) Kymographs of mitochondrial movement in cultured hippocampal neurons expressing mito-dsRED (magenta) and meGFP (green). Cells were treated for 1 h with either 4 nM AntA or vehicle and then imaged. (B) Quantification of mitochondrial motility from kymographs as in A. n = 9–13 axons per treatment from four independent animals. (C) Basal oxygen consumption (OCR) was measured in cortical neuron cultures 1 h after treatment with 4 nM AntA or vehicle. The consumption rate is normalized to cell number. n = 16–30 wells per condition from four independent animals. (D) Representative images of hippocampal neuron cell bodies expressing the fluorescent ATP:ADP ratio sensor PercevalHR. Cells were treated with 4 nM AntA or vehicle, as in A. (E) Quantification of fluorescence intensity ratio in hippocampal neurons expressing PercevalHR treated as in D. n = 11–19 cells per treatment from 3 independent animals. (F) Fibroblasts expressing PercevalHR were cultured in galactose media, treated for 1 h with 40 nM AntA or vehicle, and then imaged. In the lower two panels, 30 min after AntA addition, either glucose or galactose was added to the medium to a final concentration of 25 mM and immediately imaged. Glucose addition restored normal ATP/ADP ratios, but galactose addition did not. (G) Quantification of fibroblasts expressing PercevalHR treated as in F. n = 12–22 cells per condition from 4 independent transductions. (H) Representative image heatmaps show the degree of pixel variance as a reflection of mitochondrial movement. Fibroblasts expressing mito-dsRED, cultured in galactose media, were treated with vehicle or 40 nM AntA for 1 h. (I) Quantification of the variance in pixel intensity as an indicator of mitochondrial movement in fibroblasts treated as in H. n = 15–26 cells per condition from 4 independent animals. For all images, scale bars = 20 µm, bars on boxplots show the 10–90th percentile, and error bars on bar graphs show the SEM. P values for B, C, E, and I were calculated by two-tailed, unpaired t tests with Welch’s correction. For G, a one-way Welch ANOVA was performed with Dunnett’s T3 multiple comparison correction.

ETC inhibition decreases the ATP:ADP ratio and arrests mitochondria. (A) Kymographs of mitochondrial movement in cultured hippocampal neurons expressing mito-dsRED (magenta) and meGFP (green). Cells were treated for 1 h with either 4 nM AntA or vehicle and then imaged. (B) Quantification of mitochondrial motility from kymographs as in A. n = 9–13 axons per treatment from four independent animals. (C) Basal oxygen consumption (OCR) was measured in cortical neuron cultures 1 h after treatment with 4 nM AntA or vehicle. The consumption rate is normalized to cell number. n = 16–30 wells per condition from four independent animals. (D) Representative images of hippocampal neuron cell bodies expressing the fluorescent ATP:ADP ratio sensor PercevalHR. Cells were treated with 4 nM AntA or vehicle, as in A. (E) Quantification of fluorescence intensity ratio in hippocampal neurons expressing PercevalHR treated as in D. n = 11–19 cells per treatment from 3 independent animals. (F) Fibroblasts expressing PercevalHR were cultured in galactose media, treated for 1 h with 40 nM AntA or vehicle, and then imaged. In the lower two panels, 30 min after AntA addition, either glucose or galactose was added to the medium to a final concentration of 25 mM and immediately imaged. Glucose addition restored normal ATP/ADP ratios, but galactose addition did not. (G) Quantification of fibroblasts expressing PercevalHR treated as in F. n = 12–22 cells per condition from 4 independent transductions. (H) Representative image heatmaps show the degree of pixel variance as a reflection of mitochondrial movement. Fibroblasts expressing mito-dsRED, cultured in galactose media, were treated with vehicle or 40 nM AntA for 1 h. (I) Quantification of the variance in pixel intensity as an indicator of mitochondrial movement in fibroblasts treated as in H. n = 15–26 cells per condition from 4 independent animals. For all images, scale bars = 20 µm, bars on boxplots show the 10–90th percentile, and error bars on bar graphs show the SEM. P values for B, C, E, and I were calculated by two-tailed, unpaired t tests with Welch’s correction. For G, a one-way Welch ANOVA was performed with Dunnett’s T3 multiple comparison correction.

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