Supplementary Figure 1 Targeted disruption of the Npm gene by homologous recombination.
a, Structure of the mouse wt Npm locus (top), the targeting vector (middle) and the targeted
allele (bottom). Exons 2 to 7 are replaced by a GFP cDNA sequence in frame with exon 2. The
NEO resistance is under the control of the pGK promoter. NEO is excised through Cre-mediated
recombination between two loxP sites (triangles). Angiotensin-converting enzyme gene
promoter (pACE) drives the expression of Cre recombinase in spermatocytes, leading to deletion
of the NEO and Cre sequences in the mutant mice germ line. Position of the 5’ and 3’ probes
used to confirm the homologous recombination is shown. Primers pA, pB and pC have been used
for genotyping. E, EcoRI; S, SacI; P, PmeI; N, NcoI; Pa, PacI and A, AscI. b, Southern blot
analysis on EcoRI and SacI digested genomic DNA with 5’ and 3’ external probes demonstrates
homologous recombination in ES cells. c, Genotyping of the mutant mice by multiplex PCR on
tail genomic DNA using the indicated primers demonstrates germ line transmission and deletion
of the NEO cassette. d, Western blot analysis of Npm expression in MEFs of the indicated
genotypes. e, H&E staining of sagittal sections from wt and Npm-/- E10.5 embryos (upper
panels), and E10 embryos GFP fluorescence in cryo-sections (lower panels). f, Npm and GFP
expression in E8.5 and E9.5 wt and Npm-/- embryos. g, Npm-/- embryos have a smaller but
normally organized placenta. Comparable sections of E8.5 wt and Npm-/- embryos show
normally developed allantois (arrowheads) in the process of reaching and fusing to the corion
(arrows). h, At the interface between maternal (arrowheads) and fetal blood (arrows), embryonic
erythroblasts are reduced in number in the Npm-/- embryos. i, CD34 immunostaining of E9.5 wt
and Npm-/- embryo sections, Scale bars: 500 m in e, 50 m in i.
Supplementary Figure 2 Generation of Npm hypomorphic mice. a, Schematic representation of
the targeted hypomorphic Npm allele. A removable pGK-NEO cassette has been inserted in the
intronic sequence between exons 6 and 7 of the gene (grey ovals indicate Frt sites). Deletion of
the first half of the gene could also be obtained through Cre-mediated recombination (black
triangles indicate loxP sites). 5’ and 3’ probes used to confirm the homologous recombination
and to genotype the mutant mice are shown. E, EcoRI; P, PvuII, H, HindIII; D, DraI; Pa, PacI.
Parentheses indicate loss of the original restriction site. The lower panels show southern blot
analysis on PvuII and HindIII digested genomic DNA with 5’ and 3’ external probes
demonstrating homologous recombination in ES cells. b, The NEO cassette causes
Grisendi et al.
transcriptional interference. Npm expression in the hypomorphic series by northern blot (left
panel) and western blot (middle panel) analysis of Npm+/+, Npm+/-, Npmhy/hy and Npm-/- MEFs. c,
Wt and Npmhy/hy E13.5 embryos: similarly to Npm-/- mutants, Npmhy/hy embryos are smaller in
size, pale and generally well formed except for the anterior brain and the eye. d, Sagittal sections
of E13.5 wt and Npmhy/hy embryos show up-regulation of p53 expression in the hypomorphic
mutant (arrows). Scale bar: 250 m. Right panels show 10X magnifications of the neural tissues.
e, Ribosome profile of Npm deficient MEFs. Reduction in Npm expression levels affects the
relative amounts of the ribosomal subunit 80S in Npm+/- cells, while in Npmhy/hy cells the relative
amount of all the subunits (40S, 60S and 80S) is decreased compared to a wt controls.
Supplementary Figure 3 Analysis of apoptosis and proliferation in E9.5 wt and Npm-/- embryos.
a, Deletion of the Npm gene induces an apoptotic response with increase of activated caspase-3
expression, while Ki-67 proliferation marker is comparable in wt and mutant embryos. Scale
bars: 500 m. b, Percentages of activated caspase-3 and Ki-67 expressing cells in the
neuroepithelium of wt and Npm-/- embryos.
Supplementary Figure 4 Npm is haploinsufficient for maintenance of genomic stability and
tumour susceptibility. a, Chromosome FISH analysis of Npm deficient MEFs. Number of wt,
Npm+/- and Npmhy/hy cells with the indicated number of FISH signals per cell are shown. b,
Growth curves of wt and Npm+/- P4 MEFs in a wt and p53-/- background. Values are the average
results from 3 different MEF preparations per genotype. c, Soft agar colony forming assay on wt
and Npm+/- MEFs at the indicated passage number, concomitantly infected with RasV12 and E1A
or the control empty vectors. d, CGH Array analysis of lymphomas from Npm+/+ E-Myc and
Npm+/- E-Myc mice. Chromosomal abnormalities detected in lymphomas from Npm+/+ E-Myc
(n=4) and Npm+/- E-Myc mice (n=4) are shown. In some cases, different lymphomas from the
same mouse were analyzed, as indicated. Chromosome gains and losses are listed as numerical
abnormalities. The presence of structural abnormalities, such as deletion and duplication of
chromosome regions, is indicated for each mouse. Note that chromosome 11, where murine Npm
maps to, was never found lost or rearranged. e, LOH analysis on tail (T) and tumour (Tu) DNA
derived from Npm+/+ E-Myc and Npm+/- E-Myc mice. DNA was analyzed by southern blot
hybridization with the 5’ probe utilized to identify the Npm null allele (see also sFig. 1). Two
representative cases are shown. The Npm genotype is indicated at the bottom of the panels. f,
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Expression of the Npm protein is retained in tumors from both Npm+/+ E-Myc and Npm+/- EMyc mice as indicated by the immunohistochemical analysis of Npm in lymphomas isolated
from E-Myc mice with the indicated Npm genotype.
Supplementary Figure 5 Npm+/- mice display myelodysplastic features. a, Dyserythropoiesis in
Npm+/- mice: representative images of BM samples from Npm+/- mice, arrows point to cells with
cytoplasmic blebs and nuclear fragmentation. b, Dysplastic megakaryocites: representative
images of BM samples from one wt and one Npm+/- mouse show increased number of
megakaryocytes in Npm+/- mice (see text for details); arrows point to cells with abnormally small
hypolobated nuclei. c, Bone marrow FACS analysis summary table illustrating the number of
mice analyzed and the average values obtained (see also Fig.4d and e). d, E/M relative ratio in wt
and Npm+/- mice, evaluated as ratio between the number of erythroid and myeloid precursors in
the BM. For each mouse, 5 independent counts of total BM precursors (n=100) were performed.
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