Supplementary Materials and Methods
LAM-PCR (Supplementary figure 1)
Retroviral integration in genomic DNA was carried out by linear amplification-mediated
PCR (LAM-PCR) according to our protocol (30). After loading on agarose gel, PCR
bands were extracted (QIAquick gel extraction kit, Qiagen) and sequenced (MWG,
Germany) with specific LTR3 primer. Assignment of retroviral integration sites to mouse
chromosomal loci was obtained by blasting the NCBI database.
Southern blot (Supplementary figure 1)
BM cells from ciproxine and doxycycline treated recipient mice with leukemia were
lysed and genomic DNA was extracted by proteinase K digestion, phenol/chloroform
extraction, and ethanol precipitation. Genomic DNA (10µg) was digested with EcoRI,
which cuts once within the proviral sequence and once within the integrated locus, and
was subjected to electrophoresis and transferred onto nylon N+ membrane (GE
Healthcare). Hybridization was performed in Ultrahyb (Ambion) with 32P probes, which
were labeled by random priming according to the manufacturer instructions (GE
Healthcare). The probe consisted of a 0.5-kb fragment of the GFP gene derived from
digestion of MSCV-IRES-GFP. After stringent washes, the membrane was exposed in a
Phosphorimager (BioRad).
CGH array (Supplementary figure 2)
Molecular karyotyping was performed with high resolution (244K) Agilent Mouse CGH
Genome Array (Atlas Biolabs, Germany). Genomic DNA was extracted (Qiagen) from
leukemic cells and 3µg DNA was used for hybridization. Assignment of DNA probes to
mouse chromosomal loci was downloaded in a relational database (Microsoft Access)
and analyzed for whole chromosome gains/losses or microdeletions. Data were exported
into MeV (Multiexperiment Viewer) for analysis and were submitted to the European
Bioinformatics Institute (EMBL-EBI). ArrayExpress repository (acc. no. E-MEXP2473).
Significance Analysis of Microarrays (Supplementary figure 3)
SAM analysis was performed with MEV software to identify expressed genes commonly
dysregulated between secondary recipient mice developing leukemia.
Immunostaining (Supplementary figure 4)
For CD44 and MMP9 co-localization study, anti-CD44 PE antibody (Becton Dickinson
Biosciences) and anti-MMP9 primary antibody (ab388998, Abcam) and anti-rabbitAF488 (Invitrogen) were used for cell staining. Immunostaining with a Nikon Eclipse
TE-2000 confocal microscope (Nikon), magnification 60x.
CD44 transcription (Supplementary figure 5)
Total RNA from BM cells of ciproxine and doxycycline receiving recipient mice
suffering from leukemia was extracted from bone marrow cells (Qiagen). RNA
concentration and integrity were respectively determined by NanoDrop
spectrophotometry (NanoDrop technologies) and a Bioanalyser (Agilent technology).
Reverse transcription was obtained using Illumina TotalPrep RNA Amplification Kit
(Ambion). To determine the CD44 transcription, PCR was done with primer CD44-for;
GCACGCCATGGACAAGTT and CD44-rev; GCTGTCTTTTCAAGTTAAT.