Supplementary Figure Legends
Supplementary Figure S1. Knockout FOXM1 suppresses colony formation and
induces cellular senescence in response to DNA damage in MEFs. Clonogenic assays
were performed to assess the colony formation efficiency of Foxm1−/− MEFs and WT MEFs.
A total of 5,000 cells were seeded in six well plates, treated with 0, 20, 40, 60, 80 and 100
nM of epirubicin and grown for 15 days. The cells were then stained with crystal violet
(upper). Bars represent average ± SD from three independent experiments. Statistical
significance was determined by Student’s t-test (**P ≤ 0.01, significant; ns, non-significant).
Supplementary Figure S2. Foxm1−/− MEFs exhibited higher levels of γH2AX DNA
damage foci compared with WT MEFs after epirubicin treatment. WT and Foxm1−/−
MEFs cultured on chamber slides were either untreated (0h) or treated with 0.1 µM
epirubicin for 0.5, 2, 4, 24, 48 and 72 h. Cells were then fixed and immunostained for γH2AX
foci (green). Nuclei were counterstained with DAPI (blue). Images were acquired with Leica
TCS SP5 (63 × magnification). (B) For each time point, images of at least 100 cells were
captured and used for quantification of γH2AX foci number. The graph represents average of
three independent experiments ± S.D. Statistical analyses were conducted using Student’s ttests against the correspondent time point (***P≤0.005, significant; ns, non-significant).
Supplementary Figure S3. Quantitative analysis of FOXM1, NBS1, P-NBS1, RAD50,
MRE11, P-ATM and ATM protein expression in MCF-7 and MCF7-EpiR cells treated
with 1µM epirubicin for 0, 4, 8, 16, 24 and 48 h (from Fig. 4A). Quantitation of protein
expression was performed using ImageJ software (Image Processing and Analysis in Java).
All protein expression levels were normalised against the corresponding β-Tubulin levels.
Supplementary Figure S4. Quantitative analysis of FOXM1, NBS1, P-NBS1, RAD50,
MRE11, P-ATM, ATM, P-CHK2 and CHK2 protein expression in MCF-7 and MCF7-EpiR
cells transfected with either NS siRNA or FOXM1 siRNA and treated with 1µM
epirubicin or vehicle control for 24 h (from Fig. 4C). Quantitation of protein expression
was performed using ImageJ software (Image Processing and Analysis in Java). All protein
expression levels were normalised against the corresponding β-Tubulin levels.
Supplementary Figure S5. FOXM1 depletion abolishes P-ATM expression upon
epirubicin treatment. (A) TERT 48 human fibroblast cells were transfected with either NS
siRNA or siRNA against FOXM1. Twenty-four hours following transfection, the cells were
treated with 0.1 µM epirubicin for 0, 24 and 48 h. The protein expression profiles of FOXM1,
P-ATM, ATM, NBS1 and β-Tubulin were analysed. (B) The relative mRNA expression levels
of FOXM1, NBS1, MRE11 and RAD50 were determined using qRT-PCR (normalised
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against L19). (C) Human breast cancer cell lines of ZR-75-1 and MDA-MB-231 were
transfected with NS siRNA or siRNA against FOXM1. Following 24 h of transfection, the
mRNA expression profiles of NBS1 was analysed using qRT-PCR (normalised using L19 the
house keeping gene). Statistical significance was determined by Student’s t test (*P≤0.05,
**P ≤ 0.01, ***P≤0.005, significant; ns, non-significant).
Supplementary Figure S6. Quantitative analysis of FOXM1, NBS1, P-NBS1, RAD50,
MRE11, P-ATM, and ATM protein expression in WT and Foxm1−/− MEFs treated with
0.1 µM epirubicin for 0, 0.5, 1, 2, 4, 16 and 24 h (from Fig. 5A). Quantitation of protein
expression was performed using ImageJ software (Image Processing and Analysis in Java).
All protein expression levels were normalised against the corresponding β-Tubulin levels.
Supplementary Figure S7. Quantitative analysis of FOXM1, NBS1, P-ATM, ATM,
RAD50 and MRE11 protein expression in MCF-7 cells transfected with pcDNA3 empty
vector or pcDNA3-FOXM1 plasmids and treated with 1 µM epirubicin for 0, 6 24 and 48
h (from Fig. 6A). Quantitation of protein expression was performed using ImageJ software
(Image Processing and Analysis in Java). All protein expression levels were normalised
against the corresponding β-Tubulin levels.
Supplementary Figure S8. Reconstituting NBS1 expression restores ATM activity. The
NBS1-deficient human NBS1-LBI cells were transfected with NBS1 expression vector.
Twenty-four hours after transfection the cells were treated with 0.1 µM epirubicin, and shown
are the Western blot results obtained for P-ATM, ATM, NBS1, FOXM1 and β-Tubulin.
Supplementary Figure S9. Kaplan-Meier analysis of overall survival for NBS1 staining
with log-rank test P-values indicated. Statistical analysis of the expression patterns
revealed that there was a trend linking high NBS1 nuclear localization expression to poor
survival (p=0.164 for overall survival, log-rank test, Kaplan-Meier estimate analysis) which by
multivariate Cox regression analysis, when adjusted by patients’ T-stage and lymph-node
involvement was significantly correlated with poorer survival (RR=2.869, p=0.048).
Supplementary Figure S10. Kaplan-Meier analysis of overall survival for FOXM1 and
NBS1 mRNA transcript expression in a previously web-based published cohort (2878
breast cancer patients). High FOXM1 and NBS1 mRNA expression levels are very
significantly associated with poor survival (p<0.0001 and p=0.0012, respectively).
Supplementary Figure S11. NBS1 and FOXM1 are required for homologous
recombination repair. HR repair was assayed in HeLa cells harbouring a DR-GFP reporter
system. Cells were either transfected with control pcDNA3, pcDNA3-FOXM1 or pFlag-NBS1
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or with NS siRNA, FOXM1 siRNA or NBS1 siRNA, or with co-transfection control, pFlagNBS1 plus FOXM1 siRNA. Four-eight hours after transfection, the cells were transfected
with I-Scel plasmid. Cleavage of an I-Scel and repair by HR leads to GFP expressing cells.
The percentage of GFP positive cells was determined by FACS analysis at Day 3
posttransfection. GFP-positive cells are gated and shown in red. Experiments are performed
over 3 times in triplicates and only one set of representative samples is shown.
Supplementary Figure S12. Reconstitution of FOXM1 in Foxm1-/- MEFs abrogates the
accumulation of γH2AX foci. Foxm1-/- MEFs were transfected with either pmCherry control
(red) plasmids (A) or pmCherry-FOXM1 (red) plasmids (B) and treated with 0.1 µM of
epirubicin for 0, 0.5, 2, 4, 24, 48 and 72 h. The cells were then immunostained for
phosphorylated histone H2AX foci (green) to determine DNA double-strand breaks. Nuclei
were counterstained with DAPI (blue). γH2AX foci quantification is shown. Bars represent
the average of γH2AX foci per cell of usually a sample of >30 cells from three independent
experiments± SD. Statistical significance was determined by Student’s t-test (*P≤0.05,
***P≤0.005, significant; ns, non-significant).
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