Supplementary Materials and Methods
Comet Assays (continued)
Briefly, cells were ressuspended in 0.7% (w/v) low-melting point agarose (LMA)
(Promega, USA), kept at 37°C, and placed onto glass-slides precoated with 1%
normal-melting point agarose (Promega, USA). After a third layer of 0.7% LMA was
set, slides were immersed in cold lysis buffer (2.5 M NaCl, 100 mM Na 2EDTA, 10
mM Tris, 1% sodium sarcosinate, 250 mM NaOH (pH 10), 1% Triton X-100 and 10%
DMSO) for 1 h at 4 °C. Slides were placed in an electrophoresis tank with chilled
alkaline electrophoresis buffer (1 mM Na2EDTA, 150 mM NaOH (pH 12.8)) for 20 min
to allow DNA unwinding, followed by 15 min electrophoresis at 1.6V/cm and 300 mA.
In order to prevent occurrence of additional DNA damage, all steps described above,
were performed in the dark. Subsequently, samples were gently washed in a
neutralisation solution (400 mM Tris, pH 7.5) three times for 5 min and stained with
Vectashiled Mounting Media with DAPI (Vector Laboratories, Burlingame, CA, USA).
DNA of individual cells was visualised using the Nikon Eclipse E400 Upright
Microscope (Nikon, Kawasaki, Japan), with an excitation filter of 340-380 nm,
connected to a digital CCD camera C8484 (Hamamatsu, Japan). DNA double strand
breaks were quantified with the Comet Assay IV (Sumer Perceptive Instruments
Ltd.m Suffolk, UK) software. Pseudocolor of isolated nuclei was attributed by the
program, reflecting the condensation state of the chromatin.
γH2AX immunofluorescent staining and foci quantification (continued)
Images were collected with a Leica TCS SP5 confocal microscope (Leica
Microsystems, Mannhein, Germany) with 63x objective using LAS-AF software. For
each condition, images of at least 100 cells were captured and used for quantitative
analyses of γH2AX foci. The average number of foci in the cells for each image was
quantified using Cell Profiler software. Foci were extracted in the nuclei by a
threshold applied on the Alexa Fluor 488 signal, excluding relatively weak foci and
background.
Two-step cross-linking chromatin immunoprecipitation (continued)
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Antibody for FOXM1 (C-20) was purchased from Santa Cruz Biotechnology (Santa
Cruz, USA) and the IgG rabbit negative control from (Dako, Denmark). DNA
fragments were extracted by phenol-chloroform-isoamyl alcohol (25:24:1) and
ethanol precipitated in the presence of ammonium acetate and 60 µg glycogen.
Pellets were washed in 100% ethanol, dried, and ressuspended in 30 µL of TE.
Three microliters of DNA (10% total) were amplified in 30 PCR cycles.
2