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SUPPLEMENTARY INFORMATION
Supplementary Figure 1
FANCM controls DNA chain elongation. (A) FANCM was knocked down in the
fibroblast cell line MRC5 (shFANCM plasmid used was 1). (B) MRC5 cells
transfected with the empty vector or FANCM shRNA were pulse labeled with BrdU
for 15 minutes. The median of replication fork track length is increased in the absence
of FANCM. (C) The median of track length decreases in the absence of FANCM,
when MRC5 cells were pulse-labeled for 60 minutes. The p-values were determined
by Mann-Whitney testing.
Supplementary Figure 2
FANCM promotes constant replication fork progression. (A) Control and FANCM
shRNA expressing MRC5 cells were labeled with two consecutive pulses of 25
minutes. The track length of the first pulse (CldU) was plotted on the x-axis, while the
corresponding length of the second pulse was represented on the y-axis. The linear
regression shows a flatter angle when FANCM is depleted (right part of panel). The
correlation coefficient r drops considerably in FANCM shRNA cells. (n > 50 for each
condition). (B) Same experiment as in (A), but this time the DNA of MRC5 cells was
pulse-labeled twice for 50 minutes.
Supplementary Figure 3
FANCM opposes replication fork movement in the presence of hydroxyurea. (A)
HeLa cells were exposed to different concentrations of hydroxyurea (HU) for 60
minutes in the presence of BrdU. DNA track length of more than 100 tracks was
plotted on the y-axis for each concentration. DNA chain elongation was increased
significantly in shFANCM (1) in the presence of HU. The p-values above the graph
were generated by Mann-Whitney testing. (B) Cells were either transfected with
pSUPER-puro (control) or FANCM shRNA containing plasmid and exposed for 3
hours to 10mM HU. In cells depleted for FANCM phosphorylation of Chk1 and
H2AX are diminished. The reduction of monoubiquitination of FANCD2 in the
FANCM shRNA cells confirmed that the knockdown was efficient. (C) FANCM
shRNA cells survived better than control cells in the presence of increasing
concentrations of HU, as determined by a cell survival assay.
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Supplementary Figure 4
Cell cycle profiles and apoptotic activity of control and FANCM knockdown cells
during treatment with hydroxyurea. (A) Representative cell cycle profiles of control
and shFANCM cells treated for 0, 3, 6, and 24 hours with 2 mM hydroxyurea. Cells
were fixed with ethanol, stained with propidium iodide, sorted by FACS (FacsScan,
Becton Dickinson), and cell cycle profiles were analysed using FlowJo 8.8.6.
Doublets and debris were eliminated by standard gating. (B) At the indicated time
points, cells were lysed directly in 1x sample buffer, cell extracts were resolved by
SDS PAGE, blotted onto nitrocellulose membrane and probed with anti-FANCM,
anti-PARP1 and anti-actin antibodies. During apoptosis, PARP-1 (116 kDa) is
cleaved by caspase-3 and-7 to yield 86 kDa and 27 kDa fragments.
Supplementary Figure 5
Cell cycle profiles and apoptotic activity of control and FANCM knockdown cells
during treatment with camptothecin. (A) Representative cell cycle profiles of control
and shFANCM cells treated for 0, 3, 6, and 24 hours with 25 nM CPT. Cell cycle
profiles were obtained as described in supplementary Figure 5. (B) At the indicated
time points, apoptotic activity was evaluated by western blotting and probing with
anti-PARP1 antibody to monitor apoptosis-induced cleavage of PARP1, as described
in supplementary Figure 5.
Supplementary Figure 6
FANCM promotes progression through UV-damaged DNA. (A) DNA was damaged
by UV-C light and cells were instantly pulse labeled for 60 minutes with BrdU.
Exposure to UV-C light decreased the BrdU track length in general. The median of
track length in FANCM shRNA cells dropped by 40 % after exposure to 25J/m2 and
by 29% after 50J/m2 relative to control cells treated with the same dose of UV light.
P-values above the graph were generated by Mann-Whitney testing. (B) DNA damage
checkpoint signaling was assessed by Western Blotting. HeLa cells were released
after exposure to 50 J/m2 of UV light for up to three hours. FANCM shRNA cells
showed diminished phosphorylation of Chk1 and H2AX.
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Supplementary Figure 7
Cell cycle profiles and apoptotic activity of control and FANCM knockdown cells
during treatment with UCN01. (A) Representative cell cycle profiles of control and
shFANCM cells treated for 0, 3, 6, 12, and 24 hours with 100 nM UCN01. Cell cycle
profiles were obtained as described in supplementary Figure 5. (B) At the indicated
time points, apoptotic activity was evaluated by western blotting and probing with
anti-PARP1 antibody to monitor apoptosis-induced cleavage of PARP1, as described
in supplementary Figure 5.
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