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Mitotic progression, arrest, exit or death relies on centromere structural integrity, rather than de novo transcription

  1. Marco Novais-Cruz
  2. Maria Alba Abad
  3. Wilfred FJ van IJcken
  4. Niels Galjart
  5. A Arockia Jeyaprakash
  6. Helder Maiato  Is a corresponding author
  7. Cristina Ferrás  Is a corresponding author
  1. Universidade do Porto, Portugal
  2. University of Edinburgh, United Kingdom
  3. Erasmus Medical Center, Netherlands
Research Article
Cite this article as: eLife 2018;7:e36898 doi: 10.7554/eLife.36898
7 figures, 3 videos, 1 table, 1 data set and 1 additional file

Figures

Actinomycin D compromises SAC response.

(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).

https://doi.org/10.7554/eLife.36898.002
Figure 2 with 4 supplements
Actinomycin D compromises the localization of Aurora B and Mad1 at centromeres/kinetochores.

(A) Representative immunofluorescence images of HeLa cells treated with DMSO (NOC) or ActD for 8 hr (NOC + ActD) after nocodazole treatment with the indicated antibodies. In the NOC + ActD condition orange arrows highlight a mitotic cell with reduced levels of phosphorylated Histone H3 (H3–pS10) and devoid of the active form of Aurora B (AurB pT232). Yellow arrows highlight a mitotic cell with abolished levels of phospho H3 (H3–pS10) and devoid of total Aurora B. Scale bar = 5 µm. (B) Percentage of mitotic phospho H3-negative cells in control (NOC) and 8 hr actinomycin D (NOC + ActD)-treated HeLa cells. The bar graph represents the mean and the standard deviation from three independent experiments (NOC 0.0 ± 0.0, n = 600; NOC + ActD, 16.1 ± 4.5, n = 600; **p≤0.01 relative to control, t test) (C) Representative immunofluorescence images of Hela cells treated with DMSO (NOC) or ActD for 4 hr (NOC + ActD) after nocodazole treatment with the indicated antibodies. Scale bar = 5 µm. 10x magnification of a pair of kinetochores are shown in the right. Dashed circle encompasses a single kinetochore and the arrow indicates the position of centromeric Aurora B. Scale bar = 0.5 µm. (D) Normalized ratio of pAurora B/ACA fluorescence signal at inner centromere of NOC and NOC + ActD. Each dot represents an individual kinetochore. The red line represents the mean of all quantified kinetochores and the error bars represent the standard deviation from a pool of at least two independent experiments. (NOC, 1.00 ± 0.48, n = 474; NOC + ActD, 0.63 ± 0.23, n = 309; ****p≤0.0001 relative to control, Mann-Whitney Rank Sum Test). (E) Normalized ratio of Mad1/ACA fluorescence signal at outer kinetochores of NOC and NOC + ActD cells. Each dot represents an individual kinetochore. The red line represents the mean of all quantified kinetochores and the error bars represent the standard deviation from a pool of at least two independent experiments. (NOC, 1.00 ± 0.39, n = 261; NOC + ActD, 0.55 ± 0.39, n = 258; ****p≤0.0001 relative to control, Mann-Whitney Rank Sum Test).

https://doi.org/10.7554/eLife.36898.004
Figure 2—figure supplement 1
Actinomycin D compromise Aurora B localization in RPE1 cells.

(A) Representative immunofluorescence images of RPE1 cells treated with DMSO (NOC) or ActD for 4 hr (NOC + ActD) after nocodazole treatment with the indicated antibodies. Scale bar = 5 µm. (D) Normalized ratio of pAurora B/ACA fluorescence signal at inner centromere of NOC and NOC + ActD. Each dot represents an individual kinetochore. The red line represents the mean of all quantified kinetochores and the error bars represent the standard deviation from a pool of two technical replicates (NOC, 1.00 ± 0.42, n = 92; NOC + ActD, 0.83 ± 0.47, n = 92; *p≤0.05 relative to control, Mann-Whitney Rank Sum Test).

https://doi.org/10.7554/eLife.36898.005
Figure 2—figure supplement 2
Actinomycin D causes Aurora B displacement from centromeres onto chromosome arms.

(A) Selected time frames from fluorescence microscopy of HeLa LAP-Aurora B cells treated with DMSO, ActD, CHX or AurBi after nocodazole treatment. Images were acquired every 10 min. Scale bar = 5 µm. Time = h:min. (B) Relative fluorescence intensity curves of centromeric GFP-Aurora B for control, ActD, CHX and AurBi treated HeLa cells after nocodazole treatment. Fluorescence intensities were normalized to the level at time = 0. The curves depict mean GFP-Aurora B fluorescent intensity from analyzed cells (NOC n = 9; NOC + ActD n = 8; NOC + CHX n = 7; NOC + AurBi n = 8; from time = 0 to 30 time frames), and error bars represent the standard deviation (n.s. p>0.05, ****p≤0.0001 relative to control, Analysis of covariance). Plus symbol on the graph indicates the time when the drugs were added.

https://doi.org/10.7554/eLife.36898.006
Figure 2—figure supplement 3
Inhibition of mitotic translation or Aurora B kinase activity during prometaphase does not affect Aurora B localization.

(A) Representative immunofluorescence images of HeLa cells treated with DMSO (NOC) or CHX for 4 hr (NOC +CHX) after NOC treatment with the indicated antibodies. Scale bar = 5 µm. 10x magnification of a pair of kinetochores are shown in the right. Dashed circles encompass single kinetochores and the arrow indicate the position of centromeric Aurora B. Scale bar = 0.5 µm. (B) Normalized ratio of pAurora B/ACA fluorescence signal at centromeres of NOC and NOC + CHX treated cells. Each dot represents an individual kinetochore. The red line represents the mean of all quantified kinetochores and the error bars represent the standard deviation from a pool of two technical replicates. (NOC, 1.00 ± 0.48, n = 474; NOC + CHX, 1.09 ± 0.40, n = 116; n.s. p>0.05 relative to control, Mann-Whitney Rank Sum Test). (C) Representative immunofluorescence images of HeLa cells treated with DMSO (NOC) or Aurora B inhibitor for 4 hr (NOC + AurBi) after NOC treatment with the indicated antibodies. Scale bar = 5 µm.

https://doi.org/10.7554/eLife.36898.007
Figure 2—figure supplement 4
Actinomycin D does not compromise pMPS1 localization at kinetochores and pH3T3 levels.

(A) Representative immunofluorescence images of HeLa cells treated with DMSO (NOC) or ActD for 4 hr (NOC + ActD) after nocodazole treatment with the indicated antibodies. Scale bar = 5 µm. (B) Normalized ratio of pMPS1/ACA fluorescence signal at outer kinetochores of NOC and NOC + ActD cells. Each dot represents an individual kinetochore. The red line represents the mean of all quantified kinetochores and the error bars represent the standard deviation from a pool of two technical replicates (NOC, 1.00 ± 0.48, n = 99; NOC + ActD, 0.93 ± 0.57, n = 99; n.s. p>0.05 relative to control, Mann-Whitney Rank Sum Test). (C) Representative immunofluorescence images of HeLa cells treated with DMSO (NOC) or ActD for 4 hr (NOC + ActD) after nocodazole treatment with the indicated antibodies. Scale bar = 5 µm. (D) Normalized ratio of pH3T3/DAPI fluorescence signal of NOC and NOC + ActD cells. Each dot represents an individual cell. The red line represents the mean of all cells and the error bars represent the standard deviation from a pool of two technical replicates (NOC, 1.00 ± 0.48, n = 21; NOC + ActD, 0.82 ± 0.16, n = 22; n.s. p>0.05 relative to control, t test).

https://doi.org/10.7554/eLife.36898.008
Figure 3 with 2 supplements
Actinomycin D affect recruitment of KNL1 and pDSN1 to kinetochores.

(A) Representative immunofluorescence images of HeLa cells treated with DMSO (NOC) or ActD for 4 hr (NOC + ActD) after nocodazole treatment with the indicated antibodies. Scale bar = 5 µm. (B) Normalized ratio of pKNL1/ACA fluorescence signal at outer kinetochores of NOC and NOC + ActD cells. Each dot represents an individual kinetochore. The red line represents the mean of all quantified kinetochores and the error bars represent the standard deviation from a pool of two technical replicates (NOC, 1.00 ± 0.40, n = 126; NOC + ActD, 0.79 ± 0.35, n = 126; ****p≤0.0001 relative to control, t test). (C) Representative immunofluorescence images of Hela cells treated with DMSO (NOC) or ActD for 4 hr (NOC + ActD) after nocodazole treatment with the indicated antibodies. Scale bar = 5 µm. (D) Normalized ratio of pDNS1/ACA fluorescence signal at outer kinetochores of NOC and NOC + ActD cells. Each dot represents an individual kinetochore. The red line represents the mean of all quantified kinetochores and the error bars represent the standard deviation from a pool of two technical replicates (NOC, 1.00 ± 0.37, n = 116; NOC + ActD, 0.57 ± 0.29, n = 116; ****p≤0.0001 relative to control, Mann-Whitney Rank Sum Test). (E) Representative immunofluorescence images of HeLa cells treated with DMSO (NOC) or ActD for 4 hr (NOC + ActD) after nocodazole treatment with the indicated antibodies. Scale bar = 5 µm. (F) Normalized ratio of KNL1/ACA fluorescence signal at outer kinetochores of NOC and NOC + ActD cells. Each dot represents an individual kinetochore. The red line represents the mean of all quantified kinetochores and the error bars represent the standard deviation from a pool of two technical replicates (NOC, 1.00 ± 0.34, n = 160; NOC + ActD, 0.71 ± 0.34, n = 160; ****p≤0.0001 relative to control, Mann-Whitney Rank Sum Test). (G) Representative immunofluorescence images of HeLa cells treated with DMSO (NOC) or ActD for 4 hr (NOC + ActD) after nocodazole treatment with the indicated antibodies. Scale bar = 5 µm. (H) Normalized ratio of DSN1/ACA fluorescence signal at outer kinetochores of NOC and NOC + ActD cells. Each dot represents an individual kinetochore. The red line represents the mean of all quantified kinetochores and the error bars represent the standard deviation from a pool of two technical replicates (NOC, 1.00 ± 0.34, n = 110; NOC + ActD, 0.96 ± 0.32, n = 110; n.s. p>0.05 relative to control, t test).

https://doi.org/10.7554/eLife.36898.009
Figure 3—figure supplement 1
Actinomycin D does not affect levels of HEC1 and CENP-A (A) Representative immunofluorescence images of HeLa cells treated with DMSO (NOC) or ActD for 4 hr (NOC + ActD) after nocodazole treatment with the indicated antibodies.

Scale bar = 5 µm. (B) Normalized ratio of HEC1/ACA fluorescence signal at outer kinetochores of NOC and NOC + ActD cells. Each dot represents an individual kinetochore. The red line represents the mean of all quantified kinetochores and the error bars represent the standard deviation from a pool of two technical replicates (NOC, 1.00 ± 0.30, n = 112; NOC + ActD, 0.93 ± 0.32, n = 112; n.s. p>0.05 relative to control, Mann-Whitney Rank Sum Test). (C) Representative immunofluorescence images of HeLa cells treated with DMSO (NOC) or ActD for 4 hr (NOC + ActD) after nocodazole treatment with the indicated antibodies. Scale bar = 5 µm. (D) Normalized ratio of CENP-A/ACA fluorescence signal at outer kinetochores of NOC and NOC + ActD cells. Each dot represents an individual kinetochore. The red line represents the mean of all quantified kinetochores and the error bars represent the standard deviation from a pool of two technical replicates (NOC, 1.00 ± 0.35, n = 116; NOC + ActD, 1.04 ± 0.26, n = 116; n.s. p>0.05 relative to control, Mann-Whitney Rank Sum Test).

https://doi.org/10.7554/eLife.36898.010
Figure 3—figure supplement 2
Aurora B inhibition affects recruitment of Mad1 and KNL1 to kinetochores.

(A) Representative immunofluorescence images of HeLa cells treated with DMSO (NOC) or Aurora B inhibitor for 4 hr (NOC + AurBi) after NOC treatment with the indicated antibodies. Scale bar = 5 µm. (B) Normalized ratio of Mad1/ACA fluorescence signal at outer kinetochores of NOC and NOC + AurBi cells. Each dot represents an individual kinetochore. The red line represents the mean of all quantified kinetochores and the error bars represent the standard deviation from a pool of two technical replicates (NOC, 1.00 ± 0.36, n = 105; NOC + AurBi, 0.62 ± 0.29, n = 110; ****p≤0.0001 relative to control, Mann-Whitney Rank Sum Test). (C) Representative immunofluorescence images of HeLa cells treated with DMSO (NOC) or Aurora B inhibitor for 4 hr (NOC + AurBi) after NOC treatment with the indicated antibodies. Scale bar = 5 µm. (D) Normalized ratio of KNL1/ACA fluorescence signal at outer kinetochores of NOC and NOC + AurBi cells. Each dot represents an individual kinetochore. The red line represents the mean of all quantified kinetochores and the error bars represent the standard deviation from a pool of two technical replicates (NOC, 1.00 ± 0.34, n = 99; NOC + AurBi, 0.72 ± 0.37, n = 92; ****p≤0.0001 relative to control, t test).

https://doi.org/10.7554/eLife.36898.011
Actinomycin D treatment does not affect the levels of Aurora B or mitotic transcripts.

(A) Schematic representation of the experimental protocol used to study the effect of ActD in NOC induced mitotic arrested cells by western blot, RNA-seq and qPCR. (B) Western blot analysis of asynchronous (I), G2, and mitotic HeLa cell extracts (Mitosis) with the indicated antibodies. Mitotic extracts were derived from isolated mitotic cells treated cells with DMSO (control), ActD (ActD), cycloheximide (CHX) or Aurora B inhibitor (AurBi). α-tubulin was used as loading control. Approximate molecular weights are shown on the left. (C) Quantification of western blot is depicted in B. The bar represents the mean and the standard deviation from three independent experiments. (D) Normalized expression after RNA-seq of the indicated genes in control or ActD-treated mitotic cells. Error bars represent standard deviations from two independent experiments. (E) Normalized expression after qPCR of human alpha satellite gene in control or ActD-treated mitotic cells (NOC, 1.00 ± 0.01; NOC + ActD, 0.95 ± 0.02, median ±SD from three technical replicates, n.s. p>0.05 relative to control, Mann-Whitney Rank Sum Test). (F) Agarose gel electrophoresis of the qPCR product after amplification of cDNA, obtained from control or ActD-treated mitotic cells, with primers specific for the human alpha-satellite and GAPDH.

https://doi.org/10.7554/eLife.36898.012
Figure 5 with 1 supplement
Inhibition of transcription independently of DNA-intercalation does not affect spindle assembly checkpoint robustness nor the capacity to trigger mitotic cell death.

(A) Normalized expression of Mcl1 in asynchronous control HeLa cells or cells treated with ActD, triptolide or α-amanitin (ActD, 0.23 ± 0.02, Triptolide, 0.14 ± 0.03; α-amanitin, 1.13 ± 0.2, median ±SD from three technical replicates, n.s. p>0.05, ****p≤0.0001 relative to control, t test). (B) Scatter plot showing the duration of the mitotic arrest of control (NOC) and HeLa cells treated with Triptolide (NOC + Triptolide) after nocodazole treatment. The red line represents the mean and the error bars represent the standard deviation from a pool of at least two independent experiments. (NOC, 19.2 ± 7.0 hr, n = 386; NOC + Triptolide, 30.1 ± 14.7 hr, n = 301; median ±SD; n.s. p>0.05, Mann-Whitney Rank Sum Test) (C) Cell fate of mitotic HeLa cells treated with the same drugs as in 4B. (D) Representative immunofluorescence images of HeLa cells treated with DMSO (NOC) or Triptolide for 4 hr (NOC + Triptolide) after nocodazole treatment with the indicated antibodies. Scale bar = 5 µm. 10x magnification of a pair of kinetochores are shown in the right. Dashed circle encompasses a single kinetochore and the arrow indicates the position of centromeric Aurora B. Scale bar = 0.5 µm. (E) Normalized ratio of pAurora B/ACA fluorescence signal at inner centromere of NOC and NOC + Triptolide. Each dot represents an individual kinetochore. The red line represents the mean of all quantified kinetochores and the error bars represent the standard deviation from a pool of two independent experiments. (NOC, 1.00 ± 0.48, n = 474; NOC + Triptolide, 1.05 ± 0.00.40, n = 296; n.s. p>0.05 relative to control, Mann-Whitney Rank Sum Test) (F) Selected time frames from fluorescence microscopy of HeLa LAP-Aurora B cells treated with DMSO or Triptolide after nocodazole treatment. Images were acquired every 10 min. Scale bar = 5 µm. Time = h:min. (G) Relative fluorescence intensity curves of centromeric GFP-Aurora B for control and Triptolide-treated HeLa cells after nocodazole treatment. Fluorescence intensities were normalized to the level at time = 0. The curves depict mean GFP-Aurora B fluorescent intensity from analyzed cells (NOC n = 9; NOC + Triptolide n = 8; from time = 0 to 30 time frames), and error bars represent the standard deviation (n.s. p>0.05 relative to control, Analysis of covariance). Plus symbol on the graph indicates the time when the drug was added.

https://doi.org/10.7554/eLife.36898.013
Figure 5—figure supplement 1
RNA Pol II is not degraded with 12 hr α-amanitin treatment.

Western blot analysis of control asynchronous HeLa cells extracts or asynchronous HeLa cells treated with α-amanitin or Triptolide with the indicated antibodies. Cell lysates were resolved with 7% SDS-PAGE. α-tubulin was used as loading control. Dashed lines indicate that intervening lanes have been spliced out. Approximate molecular weights indicated on the left.

https://doi.org/10.7554/eLife.36898.014
DNA intercalation compromises SAC response by interfering with Aurora B localization.

(A) Normalized expression of Mcl1 in asynchronous control HeLa cells or cells treated with ethidium bromide (EtBr) (EtBr, 0.71 ± 0.06, median ±SD from three technical replicates, n.s. p>0.05, Mann-Whitney Rank Sum Test). (B) Scattered plot showing the duration of the mitotic arrest of control (NOC) and Hela cells treated with ethidium bromide (NOC + EtBr) after nocodazole treatment (NOC, 19.2 ± 7.0 hr, n = 386; NOC + EtBr, 18.1 ± 5.9 hr, n = 151; median ±SD; n.s. p>0.05, Mann-Whitney Rank Sum Test). The red line represents the mean and the error bars represent the standard deviation from a pool of at least two independent experiments. (C) Cell fate of mitotic HeLa cells treated with the same drugs as in 5B. (D) Representative immunofluorescence images of HeLa cells treated with DMSO (NOC) or ethidium bromide for 4 hr (NOC + EtBr) after nocodazole treatment with the indicated antibodies. Scale bar = 5 µm. 10x magnification of a pair of kinetochores are shown in the right. Dashed circle encompasses a single kinetochore and the arrow indicate the position of centromeric Aurora B. Scale bar = 0.5 µm. (E) Normalized ratio of pAurora B/ACA fluorescence signal at inner centromere of NOC and NOC + EtBr. Each dot represents an individual kinetochore. The red line represents the mean of all quantified kinetochores and the error bars represent the standard deviation. (NOC, 1.00 ± 0.48, n = 474; NOC + EtBr, 0.79 ± 0.23, n = 143; ****p≤0.0001 relative to control, Mann-Whitney Rank Sum Test). (F) Shows the Electrophoretic Mobility Shift Assay (EMSA) experiments carried out to assess the binding chromosomal passenger complex (CPC) ability to recombinant nucleosome core particles (NCPs) in the presence of DMSO, ActD, Triptolide or EtBr. Different molar concentrations (0, 25 and 50 nM) of CPC were tested. (G) The bar diagram shows the densitometric profile of the autoradiograph depicted in F. The bar represents the mean ±SD from at least three independent experiments (n.s. p>0.05, *p≤0.05 relative to control, t test).

https://doi.org/10.7554/eLife.36898.016
Figure 7 with 1 supplement
Mitotic progression and cytokinesis after nocodazole washout does not require de novo transcription.

(A) Schematic representation of the experimental protocol used to study the effect of the transcription inhibitors ActD and triptolide on mitotic progression. (B) Selected time frames from phase contrast microscopy of HeLa cells treated after the NOC washout with DMSO, ActD or Triptolide. Images were acquired every 20 min. For control (DMSO) and Triptolide (Triptolide), arrows highlight a normal mitotic progression after nocodazole washout. For ActD-treated cells (ActD), arrows highlight a cell that fails to divide after release from nocodazole arrest. Scale bar = 10 µm. Time = hr:min. (C) Scattered plot showing the mitotic duration between nocodazole washout and anaphase onset in control (DMSO), ActD and Triptolide treated HeLa cells (DMSO, 79.1 ± 28.8 min, n = 116; ActD, 140.5 ± 55.7 hr, n = 79; Triptolide, 86.2 ± 31.9 hr, n = 113, median ±SD from a pool of two technical replicates, n.s. p>0.05, ****p≤0.0001, Mann-Whitney Rank Sum Test). (D) Cell fate of mitotic HeLa cells treated with the same drugs as in 7B.

https://doi.org/10.7554/eLife.36898.017
Figure 7—figure supplement 1
Actinomycin D promotes cytokinesis failure.

(A) Representative immunofluorescence images of HeLa cells treated with DMSO, ActD or Triptolide for 2 hr after the NOC washout with the indicated antibodies. For ActD-treated cells, arrow highlights a cell with missegregated DNA in the spindle midzone. Scale bar = 5 µm. Dashed square encompasses a DNA bridge on the midzone and respective 5x magnification.

https://doi.org/10.7554/eLife.36898.018

Videos

Video 1
Phase contrast microscopy of HeLa cells treated with DMSO, Mps1 or ActD after nocodazole (NOC) treatment with (+) or without (-) MG132.

Images were acquired every 15 min. Time = h:min.

https://doi.org/10.7554/eLife.36898.003
Video 2
Phase contrast microscopy of HeLa cells treated with DMSO, Triptolide or ethidium bromide after nocodazole (NOC) treatment.

Images were acquired every 15 min. Time = h:min.

https://doi.org/10.7554/eLife.36898.015
Video 3
Phase contrast microscopy of HeLa cells treated with DMSO, ActD or Triptolide after NOC washout.

Images were acquired every 20 min. Time = hr:min.

https://doi.org/10.7554/eLife.36898.019

Tables

Key resources table
Reagent typeDesignationSource or referenceIdentifiersAdditional information
Cell line
(human)
HeLa parentalProvided by
Y. Mimori-Kiyosue
Cell line
(human)
GFP-Aurora
B HeLa
Provided by
M. Lampson
Cell line
(human)
Cyclin B1-
Venus HeLa
Provided by
J. Pines
Antibodyanti-Aurora B
pT232 (Rabbit
Polyclonal)
Rockland
Immuno
chemicals
Cat#600-401-677IF: (1:500)
Antibodyanti-Aim1
(Mouse Monoclonal)
BD BiosciencesCat#611083IF: (1:500)
WB: (1:1,000)
Antibodyanti-Mad1
(Mouse Monoclonal)
Merck MilliporeCat#MABE867IF: (1:500)
Antibodyanti-phospho-
Histone
H3 Ser10
(Mouse Monoclonal)
AbcamCat#ab14955IF: (1:100,000)
Antibodyanti-Cleaved
Caspase-3
(Rabbit Polyclonal)
Cell Signaling
Technology
Cat#9661IF: (1:1,000)
Antibodyanti-Centromere
antibody (Human)
FitzgeraldCat#90C-CS1058IF: (1:500)
Antibodyanti-phospho-
Mps1
Thr676 (Rabbit
Polyclonal)
a gift from G. KopsIF: (1:2,000)
Antibodyanti-phospho-
Histone
H3 Thr3 (Mouse
Monoclonal)
a gift from
J. Higgins
IF: (1:1,000)
Antibodyanti-HEC1
(Mouse
Monoclonal)
a gift from
R. Gassmann
IF: (1:2,000)
Antibodyanti-KNL1
(Rabbit
Polyclonal)
a gift from
R. Gassmann
IF: (1:500)
Antibodyanti-phosho
DSN1
(Rabbit
Polyclonal)
a gift from
I. Cheeseman
IF: (1:1,000)
Antibodyanti-phospho
KNL1
(Rabbit
Polyclonal)
a gift from
I. Cheeseman
IF: (1:1,000)
Antibodyanti-CENPA
(Mouse
Monoclonal)
a gift from
L. Jansen
IF: (1:200)
Antibodyanti-DSN1
(Mouse
Monoclonal)
a gift from
A. Musacchio
IF: (1:200)
AntibodyAlexa 488- or
568-or 647
secondaries
InvitrogenIF: (1:1,000)
Antibodyanti-Survivin
(Rabbit
Polyclonal)
Novus
Biologicals
Cat#NB500-201WB: (1:1,000)
Antibodyanti-Borealin
(Rabbit
Polyclonal)
a gift from
R. Gassmann
WB: (1:1,000)
Antibodyanti-α-tubulin
clone
B-512 (Mouse
Monoclonal)
Sigma-AldrichCat#T5168WB: (1:5,000)
Antibodyanti-RNA Pol
II S2
(Rabbit
Polyclonal)
AbcamCat#ab5095WB: (1:1,000)
Antibodyanti-mouse or
anti-rabbit
Jackson
Immuno
Research
WB: (1:5,000)
Chemical
compound, drug
NocodazoleSigma-AldrichCat#M14041 μM
Chemical
compound, drug
MG132EMD MilliporeCat#133407-82-65 μM
Chemical
compound, drug
Mps1-IN-1Provided
by N. Gray
10 μM
Chemical
compound, drug
RO3306RocheCat#SML056910 μM
Chemical
compound, drug
Actinomycin DSigma-AldrichCat#A94158 μM
Chemical
compound, drug
α-amanitinSigma-AldrichCat#A22632 μM
Chemical
compound, drug
TriptolideSigma-AldrichCat#T36521 μM
Chemical
compound, drug
CycloheximideSigma-AldrichCat#0181035.5 μM
Chemical
compound, drug
Ethidium
Bromide
Sigma-AldrichCat#E875125 μM
Chemical
compound, drug
ZM447439
(Aurora B
inhibitor)
Selleckchem.comCat#S11033.3 μM

Data availability

Processed RNA-seq have been deposited and can be consulted at https://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-6661

The following data sets were generated
  1. 1
    RNA seq Mitotic cells
    1. Ferrás C
    (2018)
    Publicly available at ArrayExpress (accession no. E-MTAB-6661).

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