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. 2021 Sep 30;12(10):893. doi: 10.1038/s41419-021-04190-w

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 2 — A ATP bioluminescent assay measuring relative ATP levels in cells released at indicated time points from a double-thymidine block. Asynchronized cells were used as controls. Mean ± SD; n = 3. B Analysis of the cell-cycle progression in MDA-MB-231 cells released from a double-thymidine block using p-HH3 (Phospho Histone H3 (S10)), p-AKA/B/C (Phospho-Aurora A (Thr288)/Aurora B (Thr232)/Aurora C (Thr198)), and p-PLK1 (Phospho-PLK1 (Thr210)). Asynchronized and nocodazole-synchronized mitotic cells were used as controls. C The relative ATP levels of mitotic cells were measured at the indicated time points after 100 nM alisertib treatment. Intracellular ATP levels were detected by ATP bioluminescent assay; n = 3. D Representative images of PercevalHR-expressing MDA-MB-231 cells synchronized in mitosis by nocodazole. The sensor bound to ATP (green), ADP (blue), or PercevalHR ratiometric signal is demonstrated. Scale bar, 25 µm. E PercevalHR ratio in mitotic cell cells after 2 h treatment with vehicle control (Ctrl), 100 nM alisertib (ALS), 5 μM ENMD-2076 (ENMD), or siRNA targeting Aurora-A (siAUR). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. By one-way ANOVA. F Quantification of pH-corrected PercevalHR ratiometric signal in single cells synchronized at mitosis. All samples were treated for 2 h with vehicle control (Ctrl), 100 nM BI-2536, 10 μM RO-3306, or 100 nM alisertib in a complete growth medium before imaging. ns not significant; *p < 0.05; ***p < 0.001; ****p < 0.0001. By one-way ANOVA. Mean ± SD, (n = 50: Ctrl; n = 43: B; n = 80: R; n = 35: A). G Quantification of pH-corrected PercevalHR ratiometric signal in interphase cells. All samples were treated as in Fig. 2F and analyzed by one-way ANOVA. ns not significant. Mean ± SD, (n = 35: Ctrl; n = 32: B; n = 22: R; n = 48: A). H Live imaging of PercevalHR expressing cells synchronized at mitosis. Quantification of intracellular F488/F405 (ATP/ADP) ratios in single cells incubated in DMEM with vehicle control (DMSO), BI-2536, RO-3306, or alisertib. (Lower right) Linear fitting of F488/F405 (ATP/ADP) ratios at different time points. (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.