Chromosome segregation errors: a double-edged sword - TI Pharma

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A No errors Actin Actin DAPI B DAPI Lagging chromosomes in cleavage furrow DAPI γH2AX Actin DAPI merge + actin % of telophase-cells with lagging chromosomes in furrow 40 30 20 10 Anaphase: Lagging chromosomes Telophase: Missegregating chromosomes not in furrow 0 U2OS (spontaneous) 60 40 20 0 RPE-1 (Mps1-IN-1) Figure S4. A) Left: Immunofluorescence images of RPE-1 cells acquired using DAPI (blue) to visualize the chromosomes and Phalloidin (red) to visualize the actin cytoskeleton. Right: Quantification of representative images shown on the left. The percentage of telophase cells with lagging chromosomes in the cleavage furrow was determined over all telophase cells. U2OS cells were released from a 3 hour RO block and fixed at the moment of telophase. RPE-1 cells were released from a 16 hour RO block, treated with Mps1-IN-1 and fixed at the moment of telophase. Average of 3 independent experiments is shown +/- SD. At least 100 cells were counted per experiment. B) Immunofluorescence images acquired using an antibody against gH2AX (green), DAPI (blue) and Phalloidin (red) in U2OS cells released from a 3 hour RO block and fixed at the moment of anaphase (upper panel) or telophase (lower panel). 47 Chromosome Segregation Errors cause DNA Damage 2
2 A siLuc siATM C 48 p-S1981 ATM merge w/DAPI Asynchronous Doxorubicin Monastrol release Monastrol release + ATM inh. Mps1-IN-1 p-S15 p53 70 60 50 40 30 20 10 0 % of nuclei with pS1981-ATM foci Luc ATM Monastrol release γ-H2AX siRNA B merge w/DAPI siLuc sip53 p-S15 p53 merge w/DAPI D % double positive cells 100 80 60 40 20 0 Asynchronous 60 50 40 30 20 10 0 % of nuclei with pS15-p53 foci Luc p53 Monastrol release γH2AX and pS15-p53 positive cells Doxorubicin - ATM inh. Monastrol release Figure S5. A,B) Left: RPE-1 cells were transfected with indicated siRNAs for 2 days, released from Monastrol as described before and stained for phospho-Serine-1981 on ATM (A) or phospho-Serine 15 on p53 (B) (red) and DAPI (blue) to visualize the DNA. Right: Quantification of immunofluorescence images shown in (A) and (B). n= at least 100 cells/experiment/condition. C) RPE-1 cells were treated as indicated and stained for phospho-Serine 15 on p53 (p-S15-p53) (red), gH2AX (green) and DAPI (blue). D) Quantification of immunofluorescence images shown in (C). Percentage of cells positive for both gH2AX and p-S15-p53 (double positive) was determined after treating RPE-1 cells with indicated conditions. In the Monastrol released samples (-, ATM inhibitor, AZD), only abnormal nuclei were quantified. Indicated inhibitors were added at the moment of release. For all experiments average of 3 independent experiments is shown +/- SD. n=at least 75 cells/condition. 10µM siRNA AZD
Page 2 and 3: Chromosome segregation errors: a do
Page 4 and 5: Chromosome segregation errors: a do
Page 6: Get up, stand up, stand up for your
Page 10 and 11: Chapter 1 General Introduction Anie
Page 12 and 13: cytoplasms and pinches off the memb
Page 14 and 15: which sister-chromatids are not pro
Page 16 and 17: Non-MCC members, but important mito
Page 18 and 19: completely separated. Following cle
Page 20 and 21: Two striking features of cancer cel
Page 22 and 23: predisposition at a very young age,
Page 24 and 25: that aberrant spindle formation inc
Page 26 and 27: (Fig.7C). As discussed in section 4
Page 28 and 29: Thesis outline Chromosome segregati
Page 30: 286,395,407,408 Increase (thymus) I
Page 33 and 34: 2 Abstract Various types of chromos
Page 35 and 36: 2 34 A C D E % of cells Control 100
Page 37 and 38: 2 assess whether cytokinesis induce
Page 39 and 40: 2 A Asynchronous Monastrol release
Page 41 and 42: 2 for 14 hours (unless indicated ot
Page 43 and 44: 2 Immuno Blotting Cells were lysed
Page 45 and 46: 2 Supplemental figures 44 A B C % o
Page 47: 2 A MCF7 B C 53BP1 foci/cell 46 % o
Page 51 and 52: 2 50 A B C % of EdU positive cells
Page 54 and 55: Chapter 3 Studying Chromosomal inst
Page 56 and 57: Introduction Aneuploidy is a common
Page 58 and 59: activity by ~80% 170-172 . With the
Page 60 and 61: A WT/CiMKi T649A Embryo’s: Figure
Page 62 and 63: control treated MEFs (unpaired t te
Page 64 and 65: Materials and Methods Cloning targe
Page 66 and 67: - Loop out BamHI site with site-dir
Page 68 and 69: Primers: pGH_pA_FW#2: TCTATGGCTTCTG
Page 70 and 71: was prepared using standard procedu
Page 72: 71 Cre-inducible Mps1 knock-in mous
Page 75 and 76: 4 Abstract Most of the current drug
Page 77 and 78: 4 Two other interesting mitotic tar
Page 79 and 80: 4 inhibition of the anti-apoptotic
Page 81 and 82: 4 cluster their centrosomes. This
Page 83 and 84: 4 of checkpoint function can be ach
Page 85 and 86: 4 4.5 Disadvantages of exploiting m
Page 88 and 89: Chapter 5 Elevating the frequency o
Page 90 and 91: Introduction Error-free chromosome
Page 92 and 93: Partial depletion of Mps1 or BubR1
Page 94 and 95: death is not induced in the short t
Page 96 and 97: had survived growth for 7 days in t
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The following antibodies have been
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Supplemental data Supplemental figu
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A BubR1 α-tubulin Hela-TetRBubR1 -
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- doxycycline + doxycycline - doxyc
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A B C percentage of cells % PI posi
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Abstract One of the most common hal
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clear accumulation of cells in mito
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the mice did not receive any doxycy
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113 Chromosome missegregations kill
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Chapter 6 Aneuploid tumor cells are
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Introduction Equal segregation of t
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S1A, data not shown). Based on thes
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A B U2OS + H2B-YFP percentage of ce
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tumor cells, leaving diploid cells
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A B p-S10-Histone H3 DAPI C µmol/l
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Supplemental Figure-1. Protein bioc
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Bioavailability of compound-5 after
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A B U2OS (7.5nM) Hela (10nM) RPE-1
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7 Abstract Taxanes, such as docetax
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7 of mitotic aberrations. These dat
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7 A Low CFP lifetime High CFP lifet
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7 docetaxel treatment in vitro resu
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7 chromosome unstable, which means
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7 Cells (~50.000/well) were plated
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7 Supplemental data Supplemental fi
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7 148
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8 Thesis summary Unequal separation
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8 exerted through contraction of th
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8 1.4 NoCut: preventing the inducti
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8 tumorformation using intravital i
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Addendum References Nederlandse sam
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41. McEwen, B.F., et al., CENP-E is
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2010. 6(5): p. 359-68. 124. Liu, S.
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210. Stucki, M., et al., MDC1 direc
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tumors in mice. Cancer Res, 2007. 6
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adiotherapy in breast cancer. Breas
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470. Marcus, A.I., et al., Mitotic
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559. Kienitz, A., et al., Partial d
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Nederlandse Samenvatting Celdeling,
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verdeling van tumorcellen compleet
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Publications A. Janssen, R.H.Medema
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En dan zijn er nu nog een aantal li
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promotie-stukjes! Je bent een voorb
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edankt voor al jullie hulp bij de i
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