Supplementary information:
MCF10A Plk1-depleted clones with mild depletion showed no obvious phenotype
MCF10A clones 3, 5 and 9 had modest levels of Plk1 depletion. Their growth rates were
comparable to control (Supplementary Figure 1a); their doubling time was not
significantly different from control (Supplementary Figure 1b); and moreover, the FACS
profiles of these clones were also similar to control (Supplementary Figure 1c). To
determine the proliferation status of these clones, Ki-67 immunofluorescent staining was
performed (Supplementary Figure 1d and e). Almost 100% of control and Plk1-depleted
clones showed positive Ki-67 staining, indicating that all these cells were proliferating.
These data suggest that with minimal Plk1 depletion, MCF10A cells were growing
normally and showed no obvious phenotype, consistent with our previous findings.
MCF10A clone 12 was arrested at G2/M and underwent apoptosis
MCF10A control and clone 12 were synchronized at G0 by serum starvation
(Supplementary Figure 2). After serum stimulation, control and clone12 showed similar
kinetics of G1/S progression. However, clone 12 was arrested at G2/M afterwards and
displayed a large population of apoptotic cells.
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Cells with higher Plk1 levels had some growth advantage
After Plk1 depletion, HeLa cells underwent severe cell death in the first few days.
However, a few remaining cells continued to grow and repopulated the cell population.
Plk1 western blotting showed that in these cells, Plk1 already came back to a level similar
to control (Supplementary Figure 3, 8 d post-infection). These cells were either from
uninfected cells or from cells expressing very low levels of shRNA. MCF10A cells
survived Plk1 depletion. However, Plk1 level also gradually came back in these cells,
although at a rate slower than HeLa. Therefore, in a heterogeneous population, cells with
higher Plk1 levels have some growth advantage, and would take over the population in
the long term.
Phospho-Chk2 western blotting showed that Chk2 was only modestly phosphorylated in
HeLa cells, but showed robust phosphorylation in MCF10A after Plk1 depletion
(Supplementary Figure 3). This reflects the presence of an intact checkpoint in normal
cells, but not in cancer cells. This difference could partially account for the much more
severe and rapid cell death in cancer cells after Plk1 depletion, since there is no
functional checkpoint to protect the cells from cell death before mitotic catastrophe takes
place (Castedo et al., 2004). P53 is a downstream target of Chk2. Depletion of p53 would
put normal cells into a situation similar to that of cancer cells, thus inducing cell death in
MCF10A when Plk1 was simultaneously depleted (Liu et al., 2006). As shown in
Supplementary Figure 3, the degree of Chk2 activation in MCF10A attenuated with time
and was inversely related to the extent of Plk1 depletion. Although MCF10A cells
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survive with lower Plk1 levels, sudden depletion of Plk1 may induce some stress
response and activate the checkpoint while cells try to adjust to new lower Plk1 levels. A
small sub-population of MCF10A with very severe depletion would resemble clone 12
and display checkpoint activation. However, these cells might either die or be outgrown
by cells with higher Plk1 levels, thus would be diluted with time, and the degree of
checkpoint activation also attenuated.
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References
Castedo M, Perfettini J-L, Roumier T, Yakushijin K, Horne D, Medema R et al. (2004).
The cell cycle checkpoint kinase Chk2 is a negative regulator of mitotic catastrophe.
Oncogene 23: 4353-4361.
Liu X, Lei M, Erikson RL. (2006). Normal cells, but not cancer cells, survive severe Plk1
depletion. Mol Cell Biol 26: 2093-2108.
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Figure legends:
Supplementary Figure 1 MCF10A Plk1-depleted clones with modest levels of
depletion grew normally. (a) MCF10A Plk1-depleted clones 3, 5 and 9 grew at rates
comparable to control as shown by the growth curves. (b) The doubling time of MCF10A
control and clones 3, 5 and 9. Error bars represent at least 2 independent experiments.
‘Empty Vector’ is a clone of MCF10A infected by empty lentiviral vector without any
shRNA insert. (c) The FACS profiles of asynchronous control and Plk1-depleted clones 3
and 9. (d, e) MCF10A control and Plk1-depleted clones were stained with Ki-67 antibody.
(d) Ki-67-positive cells were counted. (e) Some representative images are shown. Bars,
50μm.
Supplementary Figure 2 MCF10A clone 12 showed G2/M arrest. MCF10A control and
clone 12 were serum starved for 3 days and then stimulated with 10% horse serum. Cells
were harvested at different times after stimulation and subjected to FACS analysis.
Supplementary Figure 3 Cells with higher Plk1 levels had some growth advantage.
HeLa and MCF10A cells were infected with lentivirus targeting Plk1 and selected with
puromycin one day after infection for 2 days. The culture medium was changed on a
regular basis, dead cells were removed, and the remaining attached cells were harvested
for anti-Plk1 and anti-phospho-Chk2 western blotting at the days indicated.
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Supplementary Table 1
The percentage of G2/M (4N DNA) cells in Figure 3h
Release Time
Control (%)
Clone 12 (%)
2h
18.77
21.93
4h
21.86
22.92
6h
39.66
31.45
9h
43.83
35.94
12 h
29.02
35.19
13 h
24.44
26.97*
Supplementary Table 2
The percentage of apoptotic (less than 2N DNA) cells in Figure 3h
Release Time
Control (%)
Clone 12 (%)
9h
5.63
20.95
12 h
5.18
22.35
13 h
5.32
27.41*
* The decrease of 4N DNA cells at 13 h in clone 12 was not due to exit from mitosis, but
apoptosis.
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