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

2
Supplemental figures
44
A B
C
% of cells
Thymidine release
Thymidine release
+ Mps1-IN-1
% of cells with 53BP1 foci
100
80
60
40
20
0
100
80
60
40
20
0
Asynchronous
RO release
Doxorubicin
Normal nuclei
Abnormal nuclei
Monastrol
release
BJ-Tert cells
>5 foci
5 foci
4 foci
3 foci
2 foci
1 focus
0 foci
D E
Mps1-IN-1
(n=139)
0 50 100 150 200 250 300 350
Time (minutes) after anaphase
% of cells
100
80
60
40
20
0
53BP1 γ-H2AX merge w/DAPI
RPE-1 cells
normal
% γH2AX positive cells
abnormal
Monastrol block
100
80
60
40
20
0
normal
abnormal
1 hour 2 hours
average number of 53BP1-foci/cell
3,0
2,5
2,0
1,5
1,0
0,5
0
-
Control
+ +
Normal
Abnormal
Thymidine release
U2OS cells
Asynchronous
>5 foci
5 foci
4 foci
3 foci
2 foci
1 focus
0 foci
Mps1-IN-1
Mps1-IN-1
Figure S1.
A) Quantification of the amount of gH2AX foci per nucleus. RPE-1 cells were treated as described in Fig.1C, D. Average percentage
of cells with indicated amount of foci is shown of 3 independent experiments +/- SD. At least 100 cells were counted per condition/
experiment. B) Quantification of gH2AX immunostaining of RPE-1 cells treated with Monastrol for 1 or 2 hours. After this treatment,
mitotic cells were collected by shake-off, washed, replated and fixed 6 hours later. Normal: oval shaped nuclei with no clear aberrations.
Abnormal: cells that underwent chromosome missegregations; multilobed nuclei and/or DNA in cleavage furrow. n=at least
75 cells/condition. C) Left: Representative images of untransformed, immortalized BJ-Tert cells released from a thymidine block
treated without or with 10mM Mps1-IN-1 and harvested at t=16 hours after release. 53BP1 is shown in red and gH2AX in green.
DAPI (blue) was used to visualize DNA. Right: Quantification of images shown in left panel. Average of 3 independent experiments
is shown +/- SD, with n=100 cells per condition/per experiment. D) Timing of 53BP1 foci formation in Mps1-IN-1 treated RPE-1
cells stably expressing H2B-RFP and 53BP1-GFP. Only cells which obtained 53BP1 foci were included in this graph. N indicates the
amount of cells filmed. E) Quantification of 53BP1 foci formation using live cell imaging of U2OS cells stably expressing 53BP1-GFP.
U2OS cells were treated with or without Mps1-IN-1 and foci formation was determined within 5 hours after mitosis. Average of 4
independent experiments +/- SD is shown with n=at least 50 cells per condition/per experiment.