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. 2020 Oct 5;11:4983. doi: 10.1038/s41467-020-18769-y

Fig. 2. CDK1 activity is required for the switch-like mitochondrial hyperpolarization.

Fig. 2

a Mass-normalized TMRE traces for L1210 cells treated with 5 µM STLC (dark blue), 1 µg/ml nocodazole (light blue), or 15 µM proTAME (red) to induce mitotic arrest. TMRE signal reaches a stable but highly elevated level when mitosis is prolonged, indicative of a metabolic switch in mitosis. Boxplots show TMRE increases during mitotic arrest. Statistical significance was assessed using one-way ANOVA. b Western blot displaying CDK1 target phosphorylation levels using the MPM2 antibody. L1210 cells were treated for 30 min, with indicated chemicals prior to lysis. β-tubulin was used as a loading control. c Flow-cytometer-based quantifications of MPM2 antibody labeling. Samples were treated as in panel (b). Each dot represents a separate culture (n = 3). d Schematic indicating how the chemical inhibitors affect CDK1 activity. e Mass-normalized TMRE traces for L1210 cells treated with 5 µM STLC alone (red) or in combination with 1 µM RO-3306 (dark blue) or with 400 nM BMS-265246 (light blue) to partly inhibit CDK1. Partial inhibition of CDK1 reduces the extent of the metabolic switch in mitosis. Boxplots show TMRE increases during mitotic arrest. f Mass-normalized TMRE traces for a L1210 cell treated with 5 µM STLC. Once the cell was arrested in mitosis, 100 nM okadaic acid (yellow trace) or EtOH (control, 0.1% v/v, red trace) was injected (black arrow) to the culture media. Boxplots show TMRE levels before and after injection. g Mass-normalized TMRE trace for a L1210 cell treated with 5 µM STLC. Once the cell was arrested in mitosis, 5 µM RO-3306 (blue trace) or DMSO (control, 0.1% v/v, red trace) was injected (black arrow) to the culture media. Boxplots show TMRE levels before and after injection. Boxplots in (a, e, f, g) depict the mean (small square), median (horizontal bar), interquartile range (IQR) (box), and 1.5× IQR (whiskers). In (a, e, f, g), N represents independent experiments. Statistical significance was assessed using one-way ANOVA followed by Sidakholm test in (c, e), or paired, two-tailed Student’s t tests (f, g).