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

5
(+SEM). B) Quantification of time-lapse analysis of U2OS-TetRMps1cells as in Fig.S1C. siRNA transfections were performed 36 hours
prior to filming. Indicated taxol concentrations were added 1 hour prior to filming. C) Immuno-blots of Cenp-E, Mps1 and α-tubulin
of U2OS-TetRMps1 cells collected after time-lapse acquisition. D) Immuno-blots of Mps1 and actin of SW480 cells transfected with
empty pSuperior (control) and pSuperiorMps1 (Mps1 shRNA) and selected for 2 days with puromycin. E) Representative pictures
of colony formations of SW480 cells on day 8. SW480 cells were transfected with pSuperior (control) and pSuperiorMps1 (Mps1
shRNA) (day 0). Cells were selected with puromycin (day 1), replated in 6 wells plates (day 2) and indicated taxol concentrations
were added on day 3. F) Automated analysis of the percentage of PI positive over Hoechst positive SW480 cells on day 6 after
transfection as in (E). Bars represent the average of 4 independent experiments (+SEM). G) Quantification of time-lapse analysis
of SW480 cells as in Fig.S8B. SW480 cells were transfected with H2B-GFP (untreated) or H2B-GFP combined with empty pSuperior
(control) or pSuperiorMps1 (Mps1 shRNA). Cells were filmed 2 days after transfection and indicated taxol concentrations were
added 1 hour prior to filming. n indicates amount of cells filmed.
A
B
C
104
% of cells in mitosis
cumulative percentage of cells
that reached anaphase
ratio: duration mitotic delay
dox/no dox
100
80
60
10µM STLC
40
20
0
0 5 10 15 20 25 30 35
time in mitosis (hours)
0,8
0,7
0,6
0,5
0,4
0,3
0,2
0,1
0
U2OS-TetRMps1
HCT116-TetRMps1
LS174T-TetRMps1
HeLa-TetRMps1
HeLa-TetRBubR1
U2OS-TetRBubR1
U2OS-TetRMAD2
VH10-TetRMps1
U2OS-TetR:
Mps1 n=99
BubR1 n=66
- doxycycline
Mad2
Mps1 n=99
BubR1 n=66 + doxycycline
Mad2 n=64
Timing mitosis
U2OS-TetRMps1 U2OS-TetRBubR1
D
U2OS-TetRMad2
100
100
100
80
80
80
- dox
60
60
60
- dox + 5nM taxol
40
20
40
20
40
20
+ dox
+ dox + 5nM taxol
0
0 20 40 60 80 100 120
0
0 20 40 60 80 100 120
0
0 20 40 60 80 100 120
chromosome condensation - anaphase (min)
10 µM STLC
n=61
% of MPM2-positive cells in
4N population
50
40
30
20
10
0
U2OS-TetRMad2
1µM taxol
- dox
+ dox
0 0 5 nM taxol
day 3 day 7 day 7
Figure S9.
A) Live cell imaging (DIC) of indicated U2OS cell lines in the presence of 10mM STLC as in Fig. 1B. B) Time-lapse analysis of the
duration of mitosis from chromosome condensation until the start of anaphase. Indicated cell lines were treated with and without
dox for 2 days and 5nM taxol was added 1 hour prior to filming. At least 30 cells were filmed per condition. C) Ratio of the average
mitotic duration of dox treated cell lines divided by the average mitotic duration of control treated cell lines in the presence of
10mM STLC. At least 40 cells were filmed per condition. D) FACS analysis of U2OS-TetRMad2 cells after doxycycline addition for 3 or
7 days. 5nM taxol was added one day after doxycycline administration. After the various treatments cells were treated with 1mM
taxol for 18 hours to determine the mitotic checkpoint efficiency. The percentage of mitotic cells was measured as the fraction of
cells with 4N DNA content that were positive for MPM2.