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

A
B
C
percentage of cells
% PI positive cells
control cenp-e
siRNA
0 5 0 5 0 5 0 5 nM taxol
20
15
10
5
0
n=71
- doxycycline
0 5 0 5 0 5 0 5
+ doxycycline
(Mad2 depleted)
nM taxol
control cenp-e control cenp-e siRNA
- doxycycline + doxycycline
(Mad2 depleted)
time-lapse (H2B-GFP)
n=63
n=38
n=29
n=69
n=65
n=36
n=34
control cenp-e
D
E
relative signal
intensity CENP-E/
CREST
F time-lapse (DIC) G
U2OS-TetR-Mad2
VH10-TetR Mps1
600
500
100
100
400
80
80
300
60
60
200
40
40
100
20
0
0 5 0 5 0 5 0 5 nM taxol
20
0
0 2 0 2 nM taxol
0
control cenp-e control cenp-e siRNA - + doxycycline
- doxycycline
no segregation defects
+ doxycycline
(Mad2 depleted)
mild segregation defects (1-5)
severe segregation defects (>5)
percentage of cells
n=27
n=56
n=39
n=28
x1000 cells
1,2
0,8
0,4
0
Mad2
actin
- + doxycycline
control
cenp-e
control
cenp-e
control
cenp-e
control
cenp-e
siRNA
siRNA
CREST CENP-E merge w/DNA
- + doxycycline
VH10-TetR Mps1
day 0
day 2
day 4
day 7 day 9
- dox
+ dox
- dox + 2nM tax
+ dox + 2nM tax
control siRNA
cenp-e siRNA
Figure S10.
A) Representative pictures of colony formations of U2OS-TetRMad2 cells on day 6 treated with or without doxycycline (day 0),
control or CENP-E siRNA (day 2) and 0 or 5nM taxol (day 3). Luciferase or GAPDH siRNA were used as a control. B) Automated analysis
of the percentage of PI positive over Hoechst positive U2OS-TetRMad2 cells on day 6 after treatment as in (A). Bars represent the
average of 4 independent experiments (+SEM). C) Quantification of time-lapse analysis as in Fig.S1C. siRNA transfections were
performed 36 hours prior to filming. Indicated taxol concentrations were added 1 hour prior to filming. n represents amount of cells
filmed. D) Immunoblots of Mad2 and actin of U2OS-TetRMad2 cells treated with and without doxycycline and control or CENP-E
siRNA. E) Top: Localization of CENP-E (green) and CREST (red) in mitotic U2OS-TetRMad2 cells transfected with control or CENP-E
siRNA. Bottom: Quantification of intensity of CENP-E staining over CREST. Average is shown of 35 kinetochores +SD of 7 analyzed
cells per condition. F) Quantification of time-lapse analysis (DIC) of chromosome segregation in anaphase in VH10 cells. n indicates
amount of cells filmed. G) Growth curve of VH10-TetRMps1 cells after indicated treatments. On day 0 doxycycline was added, on
day 2 2nM of taxol was added where indicated.
105
Chromosome missegregations kill tumor cells
5