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. 2018 Aug 6;7:e36898. doi: 10.7554/eLife.36898

Figure 1. Actinomycin D compromises SAC response.

Figure 1.

(A) Schematic representation of the experimental protocol used to study the effect of actinomycin D (ActD) on mitotic cells by live cell imaging. (B) Selected time frames from phase contrast microscopy of HeLa cells treated with nocodazole and either DMSO (NOC), ActD (NOC + ActD) or ActD with MG132 (NOC + ActD + MG132). Images were acquired every 15 min. Arrows highlight examples of control (NOC) and ActD with MG132 (NOC + ActD + MG132) cells entering and dying in mitosis after a prolonged arrest. In cells treated with ActD (NOC + ActD), arrow highlights an example of a cell entering mitosis and then exiting mitosis through mitotic slippage. Scale bar = 10 µm. Time = hr:min. (C) Scattered plot showing the duration of the mitotic arrest of HeLa cells treated with nocodazole and either Mps1 inhibitor (NOC + Mps1 i) or ActD (NOC + ActD) with or without MG132. The red line represents the mean and the error bars represent the standard deviation from a pool of at least three independent experiments (NOC, 19.2 ± 7.0 hr, n = 386; NOC + MG132, 30.1 ± 14.7 hr, n = 108; NOC + Mps1 i, 5.4 ± 2.6 hr, n = 154; NOC + Mps1 i+MG132, 30.9 ± 18.1 hr, n = 150; NOC + ActD, 16.1 ± 5.6 hr, n = 301; NOC + ActD + MG132, 20.3 ± 9.4 hr, n = 65; median ±SD; **p≤0.01, ****p≤0.0001, Mann-Whitney Rank Sum Test). (D) Cell fate of mitotic HeLa cells treated with the same drugs as in 1C. (E) Selected time frames from phase contrast and fluorescence microscopy of Cyclin B1-Venus HeLa cells treated with DMSO or ActD in the presence of nocodazole. Images were acquired every 15 min. For ActD-treated cells (NOC + ActD) arrows highlight a mitotic cell showing reduction of Cyclin B1 over time. Scale bar = 10 µm. Time = hr:min. (F) Cyclin B1 degradation curves for control and ActD-treated Cyclin B1-Venus HeLa cells after nocodazole treatment with or without MG132. Fluorescence intensities were normalized to the level at time = 0. The curves depict mean Cyclin B1-Venus fluorescent intensity from all analyzed cells per condition (NOC n = 12; NOC + MG132 n = 10; NOC + ActD n = 9; NOC + ActD + MG132 n = 10; from time = 0 to 26 time frames), and error bars represent the standard deviation (*p≤0.05, **p≤0.01, ****p≤0.0001 relative to control, Analysis of covariance).