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Supporting Information
Supplemental Figures S1-S6 and Videos S1-S5
Figure S1. Aphidicolin inhibits replication fork progression in fibroblasts and
hMECs.
A dose-response analysis (a) was performed to assess the effect of aphidicolin on
proliferation of MRC5 and MCF10A cells. A concentration of 1 µg/ml was
considered to give optimal inhibition and this concentration was used to monitor rates
of replication fork migration when MRC5 and MCF10A cells cultured in 2D were
incubated in the presence of aphidicolin for 0, 20, 60 and 240 min. BrdU was added
for 20 min to monitor the rate of replication fork migration. DNA fibre spreads were
prepared from labelled cultures and Br-labelled tracks identified by immunofluorescence (b; representative micrographs are shown). Track lengths (measured
using Zeiss LSM software) were used to calculate average fork elongation rates (Fig.
1k,l; box and whisker plots; n>200). Note that this approach provides a precise
measure of fork rate without defining the proportion of active forks at the time of
adding aphidicolin that are inhibited by the drug. While both cell types show an
immediate decline in fork rate in aphidicolin treated cells, the rate of decline and loss
of active forks was more pronounced in MCF10A cells; a dramatic difference in the
number of persistently active forks is seen at 4 h after removing aphidicolin, when
MRC5 cultures have numerous short labelled forks whereas labelled fork from
MCF10A cultures are extremely rare. Note also that the very rare BrdU-labelled
tracks seen in MCF10A cultures were typically short single forks, whereas spreads
from MRC5 cultures contained numerous clusters of short BrdU-labelled tracks,
which result from fork collapse and restart under replication stress
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. Scale bar 10
µm.
Figure S2. RPA and H2AX staining after aphidicolin treatment.
Immuno-fluorescence of total RPA32 (a) and H2AX (b) was used to monitor
activation of the DNA damage response in MRC5 and MCF10A cells. Similar
patterns of labelling were seen in nuclei of these two cell types at equivalent times
after aphidicolin treatment. Scale bars 10 µm.
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Figure S3. Aphidicolin arrest is reversible in MCF10A and MCF7.
MCF7 treated with aphidicolin were able to re-enter cycle once the arrest was
removed (a). MCF10A cells were incubated in presence of aphidicolin for 4 days and
cultured for 4 days in drug-free medium. The number of micronuclei remained
unchanged in treated and untreated cells (b). To monitor the efficiency of recovery
from prolonged (4d) S phase arrest, MCF10A cultures were labelled with BrdU
before aphidicolin treatment and the appearance of discrete chromosome territories
monitored after removing the drug (c; scheme on left). As cells divide, random
mitotic segregation yields daughter cells that each contain ~1/2 the number of BrdUlabelled CTs as their mothers; this occurs during the 2nd and subsequent mitoses as
during the 1st all CTs are labelled in one strand. Hence the number of BrdU-labelled
CTs allows the number of cell division cycles performed by a cell to be inferred. Cells
were fixed at different time points, BrdU immuno-labelled (c) and the proportion of
cells with isolated CTs or fully labelled nuclei scored (d; averages of three
independent experiments are shown; n>250 cells per time point). Scale bars 10 µm.
Figure S4. Replication factories are disassembled in MCF10A arrested in S
phase.
A protocol was designed to assess the stability of replication sites to prolonged cell
cycle arrest (a). When actively proliferating, control cells (b) display typical early,
mid and late S phase patterns when stained to reveal sites containing PCNA (b, PCNA
channel). Typical fields are shown; the PCNA positive cell on the left is in early S
phase and on the right in mid S phase. The distribution of the active replication sites
labelled with PCNA is compared to the distribution of pre-labelled CTs, which in
control cells are reduced to about 5/cell by successive rounds of random mitotic
segregation (b, Cy3dUTP channel). If replication factories persist during prolonged
aphidicolin arrest, as they do during arrest for 24 h or less, sites of Cy3dUTP and
PCNA will be coincident. In fact, following arrest very few of the Cy3dUTP labelled
cells were also PCNA positive, showing that in most cases the active replication
factories were lost (c, 3 typical examples shown). One example of a PCNA positive
and Cy3dUTP positive cell is shown (c, example on right) but the sites are not
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significantly colocalised, suggesting that changes in factory structure occur during
prolonged arrest. This behaviour is however rare, with only 3% of Cy3dUTP positive
cells having PCNA foci after 96 h aphidicolin arrest (n=66). Scale bar 10 µm.
Figure S5. Behaviour of tumourigenic breast epithelial cells.
Other human mammary epithelial cells from the luminal epithelial lineage were also
subjected to prolonged S phase arrest using aphidicolin (4 days) and recovery from
arrest monitored over a further 4-6 days (a-d). BT-474 cells were resistant to
aphidicolin and recovered after drug removal as MCF10A and MCF7 cells (a).
Conversely, T-47D died in presence of aphidicolin (b). MDA-MB- 231 and MDAMB-436 cells, which are from the basal epithelial lineage, showed robust arrest but
prolonged arrest correlated with significant cell death both during arrest and recovery
(c,d).
Figure S6 Aphidicolin induces senescence in U2-OS cells.
U2-OS cells are tumorigenic and express wild type p53 at a similar level to MCF10A
cells. Like MCF10A cells, U2-OS cells do not express p16. U2-OS cells arrested
efficiently in the presence of aphidicolin (a,b) and like MCF10A, cell and nuclear size
increased during arrest (a). Flow cytometry was used to assess cell cycle structure
during aphidicolin-induced cell cycle arrest and recovery (c). During arrest, a clear
accumulation of G2/M cells was seen (c); a small gradual shift in the G2/M peak was
consistent with a slight increase in cell size during arrest. Low levels of cell death
were seen during arrest (c,d). On removing aphidicolin very high levels of cell death
were seen (c,d) and no evidence for cell proliferation was found. Scale bars 150m.
Supplemental Videos S1-S5
MCF10A and MRC5 cells were imaged for 96 h in either aphidicolin (1 g/ml)
containing full medium or medium without aphidicolin. Bright-field images of
different fields were taken at 20 min intervals and time-lapse series reconstructed as
videos. For recovery, cells were imaged for 96 h after 96 h of aphidicolin treatment. A
10x objective lens was used.
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Supplemental Video S1. MRC5 non-treated control cells.
Supplemental Video S2. MRC5 incubated in presence of aphidicolin.
Supplemental Video S3. MCF10A non-treated control cells.
Supplemental Video S4. MCF10A incubated in presence of aphidicolin.
Supplemental Video S5. MCF10A treated 96 h in aphidicolin were washed and
grown in fresh medium. Time-lapse imaging shows 24-96 h after removing
aphidicolin to monitor cell recovery.
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