Fig. 3: Mitotic kinase inhibition induces a rapid ATP loss before mitochondrial metabolism changes. | Cell Death & Disease

Fig. 3: Mitotic kinase inhibition induces a rapid ATP loss before mitochondrial metabolism changes.

From: Modulation of oxidative phosphorylation augments antineoplastic activity of mitotic aurora kinase inhibition

Fig. 3

A OCR curves depicting mitotic MDA-MB-231 cells treated with vehicle control (Ctrl), 100 nM alisertib (ALS), or 10 μM CCCP for 0 h (left) or 4 h (right). Relative fluorescent units (RFU) were measured in 40,000 cells per well. B Quantification of RFU change rates in Fig. 3A. The results are given as the mean ± SD; n = 3. ns not significant, ***p < 0.001 by one-way ANOVA. C Quantification of MitoSOX Red fluorescence intensity in nocodazole synchronized mitotic MDA-MB-231 cells by flow cytometry. Treatment for 1 h (left) or 6 h (right) with vehicle control (Ctrl), 100 nM alisertib (ALS), 5 μM ENMD-2076 (ENMD) or 10 μM CCCP. The results are given as the mean ± SD, (n = 2: 1 h; n = 3: 6 h); ns not significant; *p < 0.05; ***p < 0.001; ****p < 0.0001. By one-way ANOVA. D Quantification of relative mitochondrial potential via labeling with the JC-1 mitochondrial dye. Cells were treated as in Fig. 3C and analyzed by one-way ANOVA. Mean ± SD, n = 3. ns not significant; *p < 0.05; ***p < 0.001. E PercevalHR ratios in mitotic cells treated with vehicle control (Ctrl), 100 nM alisertib (ALS) or 5 μM ENMD-2076 (ENMD) in glucose-free and glutamine-free DMEM or complete growth medium supplied with 10 μM CCCP for 2 h. Mean ± SD, (left, n = 29: Ctrl; n = 22: ENMD; n = 26: ALS; right, n = 21: Ctrl; n = 21: ENMD; n = 10: ALS). Statistical analysis by one-way ANOVA. *p < 0.05; ***p < 0.001. F Quantification of pH-corrected PercevalHR ratiometric signals in single cells synchronized in mitosis after treatment with vehicle control (Ctrl), 100 nM BI-2536 (B), 10 μM RO-3306 (R), or 100 nM alisertib (ALS) in DMEM without glucose and glutamine or complete growth medium with 10 μM CCCP for 2 h. Mean ± SD, (left, n = 41: Ctrl; n = 44: B; n = 35: R; n = 28: A; right, n = 46: Ctrl; n = 43: B; n = 29: R; n = 42: A). The data were analyzed by one-way ANOVA. ns not significant; *p < 0.05; ***p < 0.001; ****p < 0.0001. G Live imaging of PercevalHR-expressing cells synchronized in mitosis. Quantification of intracellular F488/F405 (ATP/ADP) ratios in single cells incubated in glucose-free and glutamine-free DMEM with vehicle control (Ctrl), BI-2536, RO-3306, or alisertib treatment (The experiment was conducted on the same panel, the same DMSO group is shown in each image; The first recorded ratio was normalized to 1), n = 10. H Linear fitting of live-time PercevalHR ratiometric measurements in Fig. 3G.

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