SUPPLEMENTARY MATERIALS
Antibodies
Two rabbit polyclonal antibodies (named Sil-A and Sil-C) were generated against the Cterminal portion of NPM mutant A (Figure 1A). To generate the Sil-A polyclonal antibody, a
synthetic 18mer peptide (NH2-QEAIQDLCLAVEEVSLRK-COOH) was produced (Primm srl,
Milan, Italy), based on the predicted aminoacid sequence derived from NPM1 mutation A.
Rabbits were immunized with KLH-conjugated peptide on days 0, 21, 28, 35, 43 and
sacrificed on day 49. For Sil-C production, a synthetic peptide of 11 aminoacids (NHCOCH3CLAVEEVSLRK-COOH) was generated (Inbio Ltd, Tallin, Estonia). Sera from rabbits were
tested for reactivity against the synthetic peptide by ELISA, according to standard procedures.
Reactive sera were subjected to epitope-specific antibody purification. Briefly, 5 mg of
synthetic peptide was bound to 0.5 g CNBr-activated Sepharose 4B (GE Healthcare
Lifesciences) for 2 hours at room temperature in 0.1 M TRIS-HCl plus 0.5 M NaCl, pH 8.0.
Sera were passed through 0.45 μm filters; 10 ml of serum were loaded for each gram of resin
column. Bound antibodies were eluted with 0.1 M Glycin-HCl plus 0.5 M NaCl, pH 2.5, and
dialyzed
with
PBS.
Purified
antibodies
were
tested
by
Western
Blot
and
immunohistochemistry (Pasqualucci L et al., Blood 108:4146-4155, 2006).
The mouse anti-NPM monoclonal antibodies (clones 322 and 376), which are suitable
for detecting NPM in paraffin-embedded sections, recognize wild-type NPM and leukaemic
NPM1 mutants, as previously described (Pasqualucci L et al., Blood 108:4146-4155, 2006).
Wild-type NPM was specifically detected using a mouse monoclonal antibody (clone FC61991) recognizing only wild-type NPM (Invitrogen, Carlsbad, CA). The last antibody was
used to evaluate the quality of protein extraction (particularly nucleophosmin extraction) from
the available samples. The anti-GFP antibody was purchased from Roche (Roche Applied
Science, Indianapolis, IN).
Case population
Primary acute myeloid leukaemia (AML) cells were obtained from patients with
diagnosed AML at several Italian haematology centres. Written informed consent to examine
leukaemic samples was obtained at each participating centre, in accordance with the
Declaration of Helsinki. Either bone marrow or peripheral blood were collected from patients
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and sent to the Perugia University haematology center where samples were processed upon
arrival the day after collection. Bone marrow biopsy was also performed and analyzed.
Patients sample preparations and cell lines
Bone marrow or peripheral blood from patients with diagnosed AML were separated on
Ficoll-Hypaque. Mononuclear cells, containing leukaemic blast cells were recovered,
distributed in different microtubes, centrifuged, and dry cell pellets were analyzed immediately
or snap-frozen in liquid nitrogen and stored at -80°C for later analysis. We analyzed 213 AML
patients (106 NPMc+ and 107 NPMc- AML). One-hundred thirty-five were analyzed
prospectively, and 78 retrospectively. Inclusion criteria for data analysis were: provision of full
information on subcellular expression of NPM protein by immunohistochemistry analysis or
NPM1 gene mutational status and good sample quality, as defined by the absence of protein
degradation when membranes were probed with an anti-NPM wild-type specific antibody..
The human leukaemic cell line OCI/AML3, which carries exon-12 NPM1 mutation A
(Quentmeier H et al., Leukaemia 19:1760-1767, 2005), was used as positive control for
NPM1 mutant A protein expression. The adherent cell lines NIH3T3 and Phoenix were used
for transient expression of NPM1 mutant variants.
NPM1 mutant constructs and cell transfection
pEGFP-NPM1 mutants A, E, G, L, P and Vi2 were generated with the QuikChange Multi
Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) as previously indicated (Falini B et
al., Blood 107:4514-4523, 2006).
The NIH3T3 or the Phoenix cell line was transfected with 10 g of cDNA by the calciumphosphate precipitation method according to standard procedures. Twenty-four hours after
transfection cells were harvested and lysed directly in Laemmli sample buffer for Western Blot
analysis.
Cell lysates preparation and Western Blot analysis
Fresh or frozen dry cell pellets of 1-2 x 106 mononuclear cells isolated from either bone
marrow or peripheral blood of patients with AML were dissolved directly in 30-60 l of
Laemmli sample buffer 1X and boiled at 95°C for 5 minutes.
One to two drops of fresh full bone marrow or peripheral blood were subjected to red cell
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lysis according to standard procedure. Briefly, 50-100 l of blood were diluted 1:4 with red cell
lysis buffer and incubated rocking at room temperature (R.T.) for 10 minutes. The white blood
cell pellet was washed and dissolved directly in Laemmli buffer as specified above.
Proteins were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on a
10% acrylamide-bisacrylamide electrophoresis gel, transferred to a polyvinylidene difluoride
membrane (PVDF; Millipore, Billerica, MA), and probed with specific antibodies followed by
horseradish peroxidase-conjugated secondary antibodies (GE Healthcare Lifesciences,
Uppsala, Sweden). Importantly, for the anti-NPM mutant specific antibodies, incubation of the
membrane with primary antibody rocking overnight at 4°C was needed. Polypeptides were
visualized using enhanced chemiluminescence (ECL) or ECL-Plus western blot detection
according to the manufacturer’s instruction (GE Healthcare Lifesciences).
Immunohistochemical studies
All immunohistochemical procedures were performed blindly on paraffin sections cut
from B5-fixed/EDTA-decalcified bone marrow biopsies, as previously described (Falini B et
al., NEJM 352:254-266, 2005). Sections were subjected to microwaving/antigen retrieval in
0.1 mM EDTA, pH 8.0, and immunostained with anti-NPM monoclonal antibodies (clones 376
and 322) using the immuno–alkaline phosphatase (APAAP) technique.
Analysis of NPM1 mutations in leukaemic samples
Exon-12 NPM1 mutations had been analyzed by direct sequencing, as previously
described (Falini B et al., NEJM 352:254-266, 2005) in 42 AML patients identified as NPMc+
AML at immunohistochemistry.
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