Supplementary Material
1) NPM1 mutation analysis
For identifying NPM1 mutations, cDNA from reverse transcription was used. Exons
11 and 12 of NPM1 were amplified. The amplification mixture contained 2 µl of cDNA, 1X
PCR Buffer, 2 mM MgCl2, 200 µM of each dNTP, 1.5 U of Taq Gold DNA polymerase
(Applied
Biosystems,
Europe)
cctggacaacatttatcaaacacggta-3’)
and
and
10
pmol
of
NPM1_1112
of
the
newly-designed
NPM1_870
R1
F
(5’(5’-
tggttctcttcccaaagtggaa-3’) primers. 50 µl of the reaction mixture were subjected to the
following thermocycler steps: 95°C for 7 min, 38 cycles of 30 s at 95°C, 30 s at 60°C, 30 s
at 72°C and final extension of 7 min. All steps were carried out on a GeneAmp PCR
System 2700 (Perkin Elmer, USA). DHPLC-based screening was done using a WAVE
DNA fragment analysis system (Transgenomics Inc., San Jose, USA), through a 6.5 mm Ø
DNAsep HT® Cartridge (Transgenomics). As first step, DHPLC was conducted in partially
denaturating conditions optimized for exon-12, as recently described (Ammatuna E et al.
Clin. Chem. 51:2165-2167, 2005). Amplicons showing an heteroduplex profile were
purified (ExoSAP-IT®, Amersham Biosciences, Sweden) and sequenced directly in both
strands (Big Dye Terminator Cycled Sequencing Kit, Perkin Elmer, USA) on an automated
sequencer ABI Prism 310 (Perkin Elmer, USA). All homoduplex samples were retested
under specific conditions for exons 9, 10 and 11. This amplification step was done using
primers
NPM1_1112
R
and
the
newly-designed
NPM1_658
F
(5’-
gaaaaagcgccagtgaagaaa-3’). DHPLC runs were performed at 54°C and 55°C, as
suggested by the NavigatorTM software predicted melting curve (Ver 1.6.0)
2) Allele-specific oligonucleotide PCR (ASO-PCR)
To confirm the identity of the mutation in exon-11, an allele-specific oligonucleotide
polymerase chain reaction (ASO-PCR) was performed using the following
reaction
mixtures: 2 µl of patient cDNA and 2 µl of cDNA from two healthy controls were amplified
by 10 pmol of the mutation-specific oligonucleotide (ASO) NPM1_882Fmut (5’caaagtggaagccaaattcaggcg-3’) and by 10 pmol of normal primer (NP) NPM1_881Fwt (5’ccaaagtggaagccaaattcatcaa-3’). 10 pmol of the common reverse primer NPM1_1112R
was used for both reaction mixtures. Both reactions contained 1X PCR Buffer, 2 mM
MgCl2, 200 µM of each dNTP, 1.5 U of Taq Gold DNA polymerase (Applied Biosystems).
50 µl of each reaction mixture were run on a GeneAmp PCR System 2700 (Perkin Elmer)
at the following amplification steps: 95°C for 7 min, 35 cycles of 30 s at 95°C, 30 s at 65°C,
30 s at 72°C and final extension of 7 min.
Mutated and wild-type amplification fragments were cut from agarose gel and purified
with QIAquik® Gel Extraction Kit (Qiagen, USA) following the manufacturer’s instructions.
Reverse strands of purified PCR products were directly sequenced as described above.
3) Cell cultures and transfection procedures
NIH-3T3 murine fibroblasts were cultured in D-MEM supplemented with 10% Bovine
Calf Serum (BCS), 1% glutamine and antibiotics. For transfection purposes, cells were
seeded overnight on glass coverslips and transfected using Lipofectamine 2000
(Invitrogen, Carlsbad, CA) following the manufacturer's instructions. After 24 hours’
incubation, cells were incubated with cycloheximide (Merck Biosciences Ltd, Nottingham,
UK) 10 mg/ml for 30 minutes. Leptomycin B (Merck Biosciences), a specific CRm1
inhibitor (Fukuda M et al., Nature 390:308-311, 1997), was added at 20 ng/ml for 5 hours.
4) Immunofluorescence analysis
For fluorescence studies, NIH 3T3 cells were rinsed in PBS containing 0.1% Triton
X-100 and fixed in 4% paraformaldehyde pH 7.4 (10 minutes), washed, counterstained
with propidium iodide, mounted on Mowiol and observed with a Zeiss (Carl Zeiss, Jena,
Germany) LSM 510 confocal microscope, using a Plan Apochromat 100x/1.4 NA oil
objective. Images were collected using 488-nm (for e-GFP) and 543-nm (for propidium
iodide) laser lines for excitation. AOTF-controlled tuning of laser lines, pinhole diameters,
and light collection configuration were optimized to obtain best signal-to-noise ratio and to
avoid fluorescence crossover. LSM 510 software regulated the microscope; images were
collected and transferred to an SGI Octane workstation (Silicon Graphics, Mountain View,
CA) for further processing. Slices were reconstructed in 3D using the shadow technique or
isosurface analysis, with Imaris software (Bitplane, Zurich, Switzerland), as previously
described (Quentmeier H et al., Leukemia 19:1760-1767, 2005). The cells were cut
electronically to analyse localization of NPM mutants.
5) Immunohistochemical detection of NPM
Nucleophosmin was detected on bone marrow paraffin sections using monoclonal
antibodies directed against fixative-resistant epitopes of NPM (clones 322 and 376
produced in B. Falini’s laboratory) and C23/nucleolin (Santa Cruz, Biotechnology,
Heidelberg, Germany), as previously described (Falini B et al., N Engl J Med 352:254-266,
2005). Paraffin sections were also stained with monoclonal antibodies directed against
glycophorin, myeloperoxidase, and CD68 (all purchased from Dako Cytomation, Glostrup,
Denmark). In all instances, antibody-antigen binding was revealed using the immunoalkaline phosphatase APAAP technique (Cordell JL et al., J Histochem Cytochem 32:219229, 1984).